Tetrahydropyran nucleic acid analogs

ABSTRACT

The present disclosure describes tetrahydropyran nucleoside analogs, oligomeric compounds prepared therefrom and methods of using the oligomeric compounds. More particularly, tetrahydropyran nucleoside analogs are provided, having one or more chiral substituents, that are useful for enhancing properties of oligomeric compounds including nuclease resistance and binding affinity. In some embodiments, the oligomeric compounds provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.12/192,847, filed Aug. 15, 2008, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application Ser. No. 60/956,100 filed Aug.15, 2007, U.S. Provisional Application Ser. No. 61/021,236 filed Jan.15, 2008, U.S. Provisional Application Ser. No. 61/031,226 filed Feb.25, 2008, and U.S. Provisional Application Ser. No. 61/052,030 filed May9, 2008. Each of the above applications is herein incorporated byreference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCHEM0041USD1SEQ.txt, created on Nov. 29, 2011 which is 4 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are tetrahydropyran nucleoside analogs, oligomericcompounds prepared therefrom and methods of using the oligomericcompounds. More particularly, the tetrahydropyran nucleoside analogseach have a substituted tetrahydropyran ring replacing the naturallyoccurring pentofuranose ring. In certain embodiments, the oligomericcompounds hybridize to a portion of a target RNA resulting in loss ofnormal function of the target RNA.

BACKGROUND OF THE INVENTION

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides are routinely used forincorporation into antisense sequences to enhance one or more propertiessuch as for example affinity and nuclease resistance. One such group ofchemically modified nucleosides includes tetrahydropyran nucleosideanalogs wherein the furanose ring is replaced with a tetrahydropyranring.

The synthesis of various tetrahydropyran nucleoside analogs has beenreported in the literature, see for example: Verheggen et al., J. Med.Chem., 1995, 38, 826-835; Altmann et al., Chimia, 1996, 50, 168-176;Herdewijn et al., Bioorganic & Medicinal Chemistry Letters, 1996, 6(13), 1457-1460; Verheggen et al., Nucleosides & Nucleotides, 1996,15(1-3), 325-335; Ostrowski et al., J. Med. Chem., 1998, 41, 4343-4353;Allart et al., Tetrahedron., 1999, 55, 6527-6546; Wouters et al.,Bioorganic & Medicinal Chemistry Letters, 1999, 9, 1563-1566; Brown, etal., Drug Development Res., 2000, 49, 253-259; published PCTapplication: WO 93/25565; WO 02/18406; and WO 05/049582; U.S. Pat. Nos.5,314,893; 5,607,922; and 6,455,507.

Various tetrahydropyran nucleoside analogs have been described asmonomers and have also been incorporated into oligomeric compounds (seefor example: Published PCT application, WO 93/25565, published Dec. 23,1993; Augustyns et al. Nucleic Acids Res., 1993, 21(20), 4670-4676;Verheggen et al., J. Med. Chem., 1993, 36, 2033-2040; Van Aerschol etal., Angew. Chem. Int. Ed. Engl., 1995, 34(12), 1338-1339; Anderson etal., Tetrahedron Letters, 1996, 37(45), 8147-8150; Herdewijn et al.,Liebigs Ann., 1996, 1337-1348; De Bouvere et al., Liebigs Ann./Recueil,1997, 1453-1461; 1513-1520; Hendrix et al., Chem. Eur. J., 1997, 3(1),110-120; Hendrix et al., Chem. Eur. J., 1997, 3(9), 1513-1520; Hossainet al, J. Org. Chem., 1998, 63, 1574-1582; Allart et al., Chem. Eur. 1,1999, 5(8), 2424-2431; Boudou et al., Nucleic Acids Res., 1999, 27(6),1450-1456; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 1108-1109;Kozlov et al., J. Am. Chem. Soc., 1999, 121, 2653-2656; Kozlov et al.,J. Am. Chem. Soc., 1999, 121, 5856-5859; Pochet et al., Nucleosides &Nucleotides, 1999, 18 (4&5), 1015-1017; Vastmans et al., CollectionSymposium Series, 1999, 2, 156-160; Froeyen et al., Helvetica ChimicaActa, 2000, 83, 2153-2182; Kozlov et al., Chem. Eur. J., 2000, 6(1),151-155; Atkins et al., Parmazie, 2000, 55(8), 615-617; Lescrinier etal., Chemistry & Biology, 2000, 7, 719-731; Lescrinier et al., HelveticaChimica Acta, 2000, 83, 1291-1310; Wang et al., J. Am. Chem., 2000, 122,8595-8602; US Patent Application US 2004/0033967; Published US PatentApplication US 2008/0038745; Published and Issued U.S. Pat. No.7,276,592). DNA analogs have also been reviewed in an article (see:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854)which included a general discussion of tetrahydropyran nucleosideanalogs (under the name: hexitol nucleic acid family).

Oligomeric compounds having phosphodiester linked 3′-H tetrahydropyrannucleoside analogs (also referred to in the art as HNA—hexitol nucleicacids or 1,5-anhydrohexitol nucleic acids) have been prepared forevaluation in cell assays. The different motifs that have been evaluatedare fully modified wherein each monomer is a phosphodiester linked 3′-Htetrahydropyran nucleoside analog and gapped wherein each monomer in the3′ and 5′ external regions of the oligomeric compound are eachphosphodiester linked 3′-H tetrahydropyran nucleoside analogs and eachmonomer in the internal region is a phosphorothioate linkeddeoxyribonucleoside (see: Kang et al., Nucleic Acids Research, 2004,32(14), 4411-4419; Vandermeeren et al., 2000, 55, 655-663; Flores etal., Parasitol Res., 1999, 85, 864-866; and Hendrix et al., Chem. Eur.J, 1997, 3(9), 1513-1520).

Oligomeric compounds having phosphodiester linked 3′-OH tetrahydropyrannucleoside analogs (also referred to in the art as ANA or D-altritolnucleic acids) have been prepared and evaluated both structurally and invitro (Allart et al., Chem. Eur. J., 1999, 5(8), 2424-2431).

Chemically modified siRNA's having incorporated hexitol nucleotides(also referred to in the art as HNA nucleic acids) have been preparedand tested for silencing capacity (see: Published PCT application, WO06/047842, published May 11, 2006.

Consequently, there remains a long-felt need for agents thatspecifically regulate gene expression via antisense mechanisms.Disclosed herein are4-substituted-5-hydroxy-6-hydroxymethyl-tetrahydropyran nucleosideanalogs that are useful in the preparation of antisense compounds formodulating gene expression pathways, including those relying onmechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well asother antisense mechanisms based on target degradation or targetoccupancy. One having skill in the art, once armed with this disclosurewill be able, without undue experimentation, to identify, prepare andexploit antisense compounds for these uses.

BRIEF SUMMARY OF THE INVENTION

Tetrahydropyran nucleoside analogs, oligomeric compounds comprising thetetrahydropyran analogs and methods of using the oligomeric compoundsare provided herein. The tetrahydropyran nucleoside analogs impartenhanced properties to oligomeric compounds they are incorporated into.

The variables are defined individually in further detail herein. It isto be understood that the tetrahydropyran nucleoside analogs, oligomercompounds, and methods of use thereof provided herein include allcombinations of the embodiments disclosed and variables defined herein.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving Formula XVI:

wherein:

Bx is a heterocyclic base moiety;

T₅ is a hydroxyl protecting group;

L₁ is H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

Z₁ is O⁻ or OE₁;

Z₂ is OH, OE₁ or N(E₁)(E₂);

each E₁ and E₂ is, independently, alkyl or substituted alkyl;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄,q₅, q₆ and q₇ is methyl.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided wherein Bx is uracil, 5-methyluracil,5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, thymine,2′-thio-thymine, cytosine, 5-methylcytosine, 5-thiazolo-cytosine,5-propynyl-cytosine, adenine, guanine, 2,6-diaminopurine,1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certainembodiments, Bx is uracil, 5-methyluracil, thymine, cytosine,5-methylcytosine, 2,6-diaminopurine, adenine or guanine.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided wherein T₅ is acetyl, t-butyl, t-butoxymethyl,methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4-methoxytrityl, 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl,trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate,tosylate, triflate, trityl, monomethoxytrityl, dimethoxytrityl,trimethoxytrityl or substituted pixyl. In certain embodiments, T₅ isacetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl ordimethoxytrityl.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided wherein L₁ is F. In certain embodiments, L₁ isH.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided wherein Z₁ is O⁻ and Z₂ is OH. In certainembodiments, Z₁ is O(CH₂)₂CN, Z₂ is N[CH₂(CH₃)₂]₂ and T₅ is4,4′-dimethoxytrityl. In certain embodiments, Z₁ is O⁻ and Z₂ is OHwhich provides an H phosphonate group at the 4′ position of thetetrahydropyran nucleoside analog which can also be written as3′-O—P(═O)(H)(OH or O⁻amine⁺). In certain embodiments, Z₁ is O(CH₂)₂CN,Z₂ is N[CH₂(CH₃)₂]₂ and T₅ is 4,4′-dimethoxytrityl which provides aphosphoramidite at the 3′-position.

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVI are provided and have the configuration as illustrated inFormula XVII:

In certain embodiments, tetrahydropyran nucleoside analogs havingFormula XVII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH; Bx is uracil, 5-methyluracil, thymine, cytosine, 5-methylcytosine,2,6-diaminopurine, adenine or guanine; T₅ is 4,4′-dimethoxytrityl; Z₁ isO(CH₂)₂CN; and Z₂ is N[CH₂(CH₃)₂]₂.

In certain embodiments, oligomeric compounds are provided comprising atleast one tetrahydropyran nucleoside analog of Formula X:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S orNJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and

wherein said oligomeric compound comprises from about 8 to about 40monomer subunits linked by internucleoside linking groups and at leastone internucleoside linking group is a phosphorothioate internucleosidelinking group.

In certain embodiments, the oligomeric compounds comprise at least twotetrahydropyran nucleoside analogs of Formula X. In certain embodiments,the oligomeric compounds comprise at least two contiguoustetrahydropyran nucleoside analogs of Formula X that are linked by aphosphorothioate internucleoside linking group.

In certain embodiments, the oligomeric compounds comprise at least onetetrahydropyran nucleoside analog of Formula X and at least oneβ-D-2′-deoxyribonucleoside. In certain embodiments, the oligomericcompounds comprise at least one tetrahydropyran nucleoside analog ofFormula X that is linked to a β-D-2′-deoxyribonucleoside by aphosphorothioate internucleoside linking group.

In certain embodiments, the oligomeric compounds comprise at least oneregion of from 2 to about 5 contiguous tetrahydropyran nucleosideanalogs of Formula X. In certain embodiments, the oligomeric compoundscomprise at least one region of from 2 to about 5 contiguoustetrahydropyran nucleoside analogs of Formula X and at least oneadditional region of from 1 to about 5 contiguous monomer subunits otherthan β-D-ribonucleosides and β-D-2′-deoxyribonucleosides wherein theadditional region is separated from the at least one region by at leastone β-D-2′-deoxyribonucleoside. In certain embodiments, oligomericcompounds are provided comprising at least two regions, each regionhaving from 1 to about 5 contiguous tetrahydropyran nucleoside analogsof Formula X and wherein the two regions are separated by at least onemonomer subunit wherein each monomer subunit is, independently, anucleoside or a modified nucleoside.

In certain embodiments, oligomeric compounds are provided comprising agapped oligomeric compounds comprising at least two regions, each regionhaving from 1 to about 5 contiguous tetrahydropyran nucleoside analogsof Formula X wherein one of said at least two regions of contiguoustetrahydropyran nucleoside analogs of Formula X is located at the 5′-endand the other of said at least two regions of contiguous tetrahydropyrannucleoside analogs of Formula X is located at the 3′-end and wherein thetwo regions are separated by an internal region comprising from about 6to about 18 monomer subunits wherein each monomer subunit is,independently, a nucleoside or a modified nucleoside.

In certain embodiments, the oligomeric compounds comprise at least onephosphodiester internucleoside linking group. In certain embodiments,each internucleoside linking group is a phosphorothioate internucleosidelinking group.

In certain embodiments, oligomeric compounds are provided wherein eachq₁, q₂, q₃, q₄, q₅, q₆ and q₇ is H. In certain embodiments, at least oneof q₁, q₂, q₃, q₄, q₅, q₆ or q₇ is other than H. In certain embodiments,at least one of q₁, q₂, q₃, q₄, q₅, q₆ or q₇ is methyl.

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has the configuration of Formula XI:

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula XII:

In certain embodiments, oligomeric compounds are provided comprisingfrom about 10 to about 21 monomer subunits in length. In certainembodiments, oligomeric compounds are provided comprising from about 10to about 16 monomer subunits in length. In certain embodiments,oligomeric compounds are provided comprising from about 10 to about 14monomer subunits in length. In certain embodiments, the comprising termis included solely to provide for additional substituent groupsroutinely added to oligomeric compounds such as but not limited toprotecting groups such as hydroxyl protecting groups, optionally linkedconjugate groups, 5′ and/or 3′-terminal groups and/or other substituentgroups.

In certain embodiments, oligomeric compounds are provided comprising atleast two tetrahydropyran nucleoside analogs of Formula XIII:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula XIII:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S orNJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

wherein said oligomeric compound comprises from about 8 to about 40monomer subunits; and

at least two of the tetrahydropyran nucleoside analogs of Formula XIIIare linked by a phosphorothioate internucleoside linking group.

In certain embodiments, oligomeric compounds are provided wherein one ofR₃ and R₄ is H and the other of R₃ and R₄ is H, OCH₃ or F for at leastone tetrahydropyran nucleoside analog of Formula XIII.

In certain embodiments, oligomeric compounds are provided comprising atleast one β-D-2′-deoxyribonucleoside. In certain embodiments, oligomericcompounds are provided comprising at least oneβ-D-2′-deoxyribonucleoside wherein at least oneβ-D-2′-deoxyribonucleoside is linked to a tetrahydropyran nucleosideanalog of Formula XIII by a phosphorothioate internucleoside linkinggroup.

In certain embodiments, oligomeric compounds are provided comprising atleast one region of from 2 to about 5 contiguous tetrahydropyrannucleoside analogs of Formula XIII. In certain embodiments, oligomericcompounds are provided comprising at least one region of from 2 to about5 contiguous tetrahydropyran nucleoside analogs of Formula XIII and atleast one additional region of from 1 to about 5 contiguous monomersubunits other than β-D-ribonucleosides or β-D-2′-deoxyribonucleosideswherein the additional region is separated from the at least one regionby at least one β-D-2′-deoxyribonucleoside. In certain embodiments,oligomeric compounds are provided comprising at least one region of from2 to about 5 contiguous tetrahydropyran nucleoside analogs of FormulaXIII and at least one additional region of from 1 to about 5 contiguoustetrahydropyran nucleoside analogs of Formula XIII wherein the at leastone region of from 2 to about 5 contiguous tetrahydropyran nucleosideanalogs of Formula XIII is separated from the additional region of from1 to about 5 contiguous tetrahydropyran nucleoside analogs of FormulaXIII by at least one nucleoside or modified nucleoside.

In certain embodiments, oligomeric compounds are provided comprising atleast two regions of from 1 to about 5 contiguous tetrahydropyrannucleoside analogs of Formula XIII comprising a gapped oligomericcompound wherein one of said at least two regions of contiguoustetrahydropyran nucleoside analogs of Formula XIII is located at the5′-end and the other of said at least two regions of contiguoustetrahydropyran nucleoside analogs of Formula XIII is located at the3′-end and wherein the two regions are separated by an internal regioncomprising from about 6 to about 14 monomer subunits wherein eachmonomer subunit is, independently, a nucleoside or a modifiednucleoside.

In certain embodiments, oligomeric compound are provided comprising atleast one phosphodiester internucleoside linking group. In certainembodiments, oligomeric compound are provided wherein eachinternucleoside linking group is a phosphorothioate internucleosidelinking group.

In certain embodiments, oligomeric compounds are provided wherein eachq₁, q₂, q₃, q₄, q₅, q₆ and q₇ is H. In certain embodiments, wherein atleast one of q₁, q₂, q₃, q₄, q₅, q₆ or q₇ is other than H. In certainembodiments, wherein at least one of q₁, q₂, q₃, q₄, q₅, q₆ or q₇ ismethyl.

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog of Formula XIII has the configurationof Formula XIV:

In certain embodiments, oligomeric compounds are provided wherein atleast one tetrahydropyran nucleoside analog has Formula XV:

wherein:

Bx is a heterocyclic base moiety; and

R₅ is H, OCH₃ or F.

In certain embodiments, oligomeric compounds are provided eachtetrahydropyran nucleoside analog has Formula XV. In certainembodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula XV and each R₅ is H. Incertain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula XV and each R₅ is OCH₃. Incertain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula XV and each R₅ is F.

In certain embodiments, oligomeric compounds are provided comprisingfrom about 10 to about 21 monomer subunits in length. In certainembodiments, oligomeric compounds are provided comprising from about 10to about 16 monomer subunits in length. In certain embodiments,oligomeric compounds are provided comprising from about 10 to about 14monomer subunits in length. In certain embodiments, the comprising termis included solely to provide for additional substituent groupsroutinely added to oligomeric compounds such as but not limited toprotecting groups such as hydroxyl protecting groups, optionally linkedconjugate groups, 5′ and/or 3′-terminal groups and/or other substituentgroups.

In certain embodiments, methods are provided comprising contacting acell in an animal with one or more oligomeric compounds provided herein.In certain embodiments, the cell is in a human. In certain embodiments,the methods are performed with an oligomeric compound provided hereinthat is complementary to a target RNA. In certain embodiments, thetarget RNA is selected from mRNA, pre-mRNA and micro RNA. In certainembodiments, the target RNA is mRNA. In certain embodiments, the targetRNA is human mRNA. In certain embodiments, the target RNA is cleavedthereby inhibiting its function.

In certain embodiments, the methods provided herein further compriseevaluating the activity of the oligomeric compound on the cell. Incertain embodiments, the step of evaluating comprises detecting thelevels of target RNA. In certain embodiments, the step of evaluatingcomprises detecting the levels of a protein. In certain embodiments, thestep of evaluating comprises detection of one or more phenotypiceffects.

In certain embodiments, tetrahydropyran nucleoside analogs of Formula Iare provided:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, the other of R₁ and R₂ is H. In certainembodiments, R₁ and R₂ are each fluoro. In certain embodiments, theother of R₁ and R₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certainembodiments, the other of R₁ and R₂ is methyl, ethyl, substituted methylor substituted ethyl. the other of R₁ and R₂ is methyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments,at least one of q₁ and q₂ is methyl. In certain embodiments, at leastone of q₃ and q₄ is methyl. In certain embodiments, at least one of q₅,q₆ and q₇ is methyl.

In certain embodiments, T₁ and T₂ are each H. In certain embodiments, atleast one of T₁ and T₂ is a hydroxyl protecting group. In certainembodiments, each hydroxyl protecting group is, independently, acetyl,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl,2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4-methoxytrityl, 4,4′-dimethoxytrityl, trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl,trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triflate, trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl or substituted pixyl.

In certain embodiments, T₁ is acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, 4-methoxytrityl or 4,4′-dimethoxytrityl. Incertain embodiments, one of T₁ and T₂ is a hydroxyl protecting group andthe other of T₁ and T₂ is diisopropylcyanoethoxy phosphoramidite orH-phosphonate. In certain embodiments, T₁ is 4,4′-dimethoxytrityl and T₂is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, Bx is uracil, thymine, cytosine, adenine orguanine. In certain embodiments, Bx is a pyrimidine, substitutedpyrimidine, purine or substituted purine wherein said substitution isother than an intercalator or a linked group that does not interact witha nucleic acid target when the tetrahydropyran nucleoside analog islocated in an oligomeric compound. In certain embodiments, Bx is uracil,5-methyluracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil,thymine, 2′-thio-thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine,2,6-diaminopurine, 1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one. Bx is uracil,5-methyluracil, 5-propynyl-uracil, thymine, cytosine, 5-methylcytosine,5-propynyl-cytosine, adenine or guanine.

In certain embodiments, the tetrahydropyran nucleoside analogs have theconfiguration shown in Formula Ia:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving the configuration shown in formula Ia wherein R₂ is fluoro. Incertain embodiments, tetrahydropyran nucleoside analogs are providedhaving the configuration shown in formula Ia wherein R₁ is H and R₂ isfluoro. In certain embodiments, tetrahydropyran nucleoside analogs areprovided having the configuration shown in formula Ia wherein R₁ is H,R₂ is fluoro and q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving the configuration shown in formula Ia wherein R₁ is C₁-C₆ alkylor substituted C₁-C₆ alkyl. In certain embodiments, tetrahydropyrannucleoside analogs are provided having the configuration shown informula Ia wherein R₁ is methyl, ethyl, substituted methyl orsubstituted ethyl. In certain embodiments, tetrahydropyran nucleosideanalogs are provided having the configuration shown in formula Iawherein R₁ and R₂ are each fluoro.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving Formula II:

wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds comprising at least onetetrahydropyran nucleoside analog of Formula III are provided:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula III:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein said oligomeric compound comprises from about 8 to about 40monomeric subunits.

In certain embodiments, the other of R₁ and R₂ is H. In certainembodiments, R₁ and R₂ are each fluoro. In certain embodiments, theother of R₁ and R₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certainembodiments, the other of R₁ and R₂ is methyl, ethyl, substituted methylor substituted ethyl. In certain embodiments, the other of R₁ and R₂ ismethyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments,at least one of q₁ and q₂ is methyl. In certain embodiments, at leastone of q₃ and q₄ is methyl. In certain embodiments, at least one of q₅,q₆ and q₇ is methyl.

In certain embodiments, at least one of T₃ and T₄ is a linked conjugategroup.

In certain embodiments, each internucleoside linking group is,independently, a phosphodiester or a phosphorothioate internucleosidelinking group. In certain embodiments, each internucleoside linkinggroup is a phosphorothioate internucleoside linking group.

In certain embodiments, each Bx is, independently, uracil, thymine,cytosine, adenine or guanine. In certain embodiments, each Bx is,independently, a pyrimidine, substituted pyrimidine, purine orsubstituted purine wherein said substitution is other than anintercalator or a linked group that does not interact with a nucleicacid target. In certain embodiments, Bx is, independently, uracil,5-methyluracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil,thymine, 2′-thio-thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine,2,6-diaminopurine, 1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certainembodiments, each Bx is, independently, uracil, 5-methyluracil,5-propynyl-uracil, thymine, cytosine, 5-methylcytosine,5-propynyl-cytosine, adenine or guanine.

In certain embodiments, each tetrahydropyran nucleoside analog ofFormula III has the configuration shown in Formula IIIa:

wherein

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving Formula IIIa wherein R₂ is fluoro. In certain embodiments,tetrahydropyran nucleoside analogs are provided having Formula IIIawherein R₂ is fluoro and R₁ is H. In certain embodiments,tetrahydropyran nucleoside analogs are provided having Formula IIIawherein R₂ is fluoro, R₁ is H and q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH.

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula IV:

wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds are provided having atleast one contiguous region of from 1 to about 5 tetrahydropyrannucleoside analogs wherein each tetrahydropyran nucleoside analog hasFormula IIIa. In certain embodiments, oligomeric compounds are providedhaving at least one contiguous region of from 1 to about 5tetrahydropyran nucleoside analogs wherein each tetrahydropyrannucleoside analog has Formula IIIa and the oligomeric compound comprisesa blockmer. In certain embodiments, oligomeric compounds are providedhaving at least one contiguous region of from 1 to about 5tetrahydropyran nucleoside analogs wherein each tetrahydropyrannucleoside analog has Formula IIIa and the oligomeric compound comprisesa 3′ or 5′-hemimer.

In certain embodiments, oligomeric compounds are provided having atleast one contiguous region of from 1 to about 5 tetrahydropyrannucleoside analogs wherein each tetrahydropyran nucleoside analog hasFormula IV:

wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds are provided having atleast two regions of from 1 to about 5 contiguous tetrahydropyrannucleoside analogs having Formula Ma that are separated by at least onenucleoside or modified nucleoside. In certain embodiments, oligomericcompounds are provided having at least two regions of from 1 to about 5contiguous tetrahydropyran nucleoside analogs having Formula IIIacomprising a gapped oligomeric compound wherein one of said at least tworegions of tetrahydropyran nucleoside analogs is located at the 5′-endand the other region of said at least two regions of tetrahydropyrannucleoside analogs is located at the 3′-end and wherein the two regionsof tetrahydropyran nucleoside analogs are separated by an internalregion comprising from about 6 to about 14 monomeric subunitsindependently selected from nucleosides, modified nucleosides andtetrahydropyran nucleoside analogs. In certain embodiments, essentiallyeach monomeric subunit in the internal region is aβ-D-2′-deoxyribonucleoside. In certain embodiments, the internal regioncomprises from about 6 to about 14 β-D-2′-deoxyribonucleosides. Incertain embodiments, the internal region comprises from about 10 toabout 12 β-D-2′-deoxyribonucleosides. In certain embodiments, theinternal region comprises from about 10 to about 14β-D-2′-deoxyribonucleosides.

In certain embodiments, oligomeric compounds are provided having atleast two regions of from about 2 to about 3 contiguous tetrahydropyrannucleoside analogs having Formula IIIa comprising a gapped oligomericcompound wherein one of said at least two regions of tetrahydropyrannucleoside analogs is located at the 5′-end and the other region of saidat least two regions of tetrahydropyran nucleoside analogs is located atthe 3′-end and wherein the two regions of tetrahydropyran nucleosideanalogs are separated by an internal region comprising from about 6 toabout 14 monomeric subunits independently selected from nucleosides,modified nucleosides and tetrahydropyran nucleoside analogs. In certainembodiments, each region of tetrahydropyran nucleoside analogsindependently comprises 2 tetrahydropyran nucleoside analogs. In certainembodiments, each region of tetrahydropyran nucleoside analogsindependently comprises 2 tetrahydropyran nucleoside analogs and theinternal region comprises 10 β-D-2′-deoxyribonucleosides.

In certain embodiments, gapped oligomeric compounds are provided whereineach region of tetrahydropyran nucleoside analogs independentlycomprises 2 tetrahydropyran nucleoside analogs and the internal regioncomprises 10 β-D-2′-deoxyribonucleosides and each tetrahydropyrannucleoside analog has Formula IV:

wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds are provided having atleast two regions of from about 2 to about 3 contiguous tetrahydropyrannucleoside analogs having Formula IIIa comprising a gapped oligomericcompound wherein one of said at least two regions of tetrahydropyrannucleoside analogs is located at the 5′-end and the other region of saidat least two regions of tetrahydropyran nucleoside analogs is located atthe 3′-end and wherein the two regions of tetrahydropyran nucleosideanalogs are separated by an internal region comprising 14β-D-2′-deoxyribonucleosides. In certain embodiments, each region oftetrahydropyran nucleoside analogs independently comprises 2tetrahydropyran nucleoside analogs. In certain embodiments, eachtetrahydropyran nucleoside analog has Formula IV:

wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, gapped oligomeric compounds are provided furthercomprising a 3′-terminal group. In certain embodiments, the 3′-terminalgroup comprises from 1 to about 4 modified or unmodified nucleosides.

In certain embodiments, oligomeric compounds are provided comprisingfrom about 10 to about 21 monomer subunits in length. In certainembodiments, oligomeric compounds are provided comprising from about 10to about 16 monomer subunits in length. In certain embodiments,oligomeric compounds are provided comprising from about 10 to about 14monomer subunits in length.

In certain embodiments, oligomeric compounds comprising at least twocontiguous tetrahydropyran nucleoside analogs of Formula V are provided:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁;

said oligomeric compound comprises from about 8 to about 40 monomericsubunits; and

wherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group.

In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments,at least one of q₁ and q₂ is methyl. In certain embodiments, at leastone of q₃ and q₄ is methyl. In certain embodiments, at least one of q₅,q₆ and q₇ is methyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H and R₃ isH. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃is H and R₄ is H. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇are each H, R₃ is H and R₄ is OCH₃. In certain embodiments, q₁, q₂, q₃,q₄, q₅, q₆ and q₇ are each H, R₃ is H and R₄ is fluoro. In certainembodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is H and R₄ ishydroxyl. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH, R₃ is H and each R₄ is H, OCH₃, fluoro or hydroxyl.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least one of T₃ and T₄ is a linked conjugate group andwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group. In certainembodiments, at least two of said at least two contiguoustetrahydropyran nucleoside analogs are linked by a phosphorothioateinternucleoside linking group. In certain embodiments, at least two ofsaid at least two contiguous tetrahydropyran nucleoside analogs arelinked by a phosphorus containing internucleoside linking group. Incertain embodiments, at least two of said at least two contiguoustetrahydropyran nucleoside analogs are linked by a non phosphoruscontaining internucleoside linking group. In certain embodiments, atleast two of said at least two contiguous tetrahydropyran nucleosideanalogs are linked by a neutral internucleoside linking group. Incertain embodiments, each internucleoside linking group is independentlya phosphodiester or a phosphorothioate internucleoside linking group. Incertain embodiments, each internucleoside linking group is aphosphorothioate internucleoside linking group.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereineach Bx is, independently, uracil, thymine, cytosine, adenine orguanine. In certain embodiments, each Bx is, independently, apyrimidine, substituted pyrimidine, purine or substituted purine whereinsaid substitution is other than an intercalator or a linked group thatdoes not interact with a nucleic acid target. In certain embodiments,each Bx is, independently, uracil, 5-methyluracil, 5-thiazolo-uracil,2-thio-uracil, 5-propynyl-uracil, thymine, 2′-thio-thymine, cytosine,5-methylcytosine, 5-thiazolocytosine, 5-propynyl-cytosine, adenine,guanine, 2,6-diaminopurine, 1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]-pyrrolo[2,3-d]pyrimidin-2-one. In certainembodiments, each Bx is, independently, uracil, 5-methyluracil,5-propynyl-uracil, thymine, cytosine, 5-methylcytosine,5-propynyl-cytosine, adenine or guanine.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereineach tetrahydropyran nucleoside analog of Formula V has theconfiguration shown in formula Va:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy; and

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vawherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereinthe oligomeric compound comprises at least one contiguous region of from1 to about 5 tetrahydropyran nucleoside analogs. In certain embodiments,the oligomeric compound comprises a blockmer. In certain embodiments,the oligomeric compound comprises a 3′ or 5′-hemimer.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vawherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereinthe oligomeric compound comprises at least two regions of from 1 toabout 5 contiguous tetrahydropyran nucleoside analogs that are separatedby at least one nucleoside or modified nucleoside. In certainembodiments, the oligomeric compound comprises a gapped oligomericcompound wherein one external region of tetrahydropyran nucleosideanalogs is located at the 5′-end and a second external region oftetrahydropyran nucleoside analogs is located at the 3′-end wherein thetwo external regions are separated by an internal region comprising fromabout 6 to about 14 monomeric subunits independently selected fromnucleosides, modified nucleosides and tetrahydropyran nucleosideanalogs. In certain embodiments, essentially each monomeric subunit inthe internal region is a β-D-2′-deoxyribonucleoside. In certainembodiments, the internal region comprises from about 6 to about 14β-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises from about 10 to about 12 β-D-2′-deoxyribonucleosides. Incertain embodiments, the internal region comprises from about 10 toabout 14 β-D-2′-deoxyribonucleosides. In certain embodiments, eachexternal region independently comprises from 2 to about 3tetrahydropyran nucleoside analogs. In certain embodiments, eachexternal region independently comprises 2 tetrahydropyran nucleosideanalogs. In certain embodiments, each external region independentlycomprises 2 tetrahydropyran nucleoside analogs and the internal regioncomprises 10 β-D-2′-deoxyribonucleosides.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereineach tetrahydropyran nucleoside analog has the Formula and configurationshown in Formula Vb:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and

R₄ is H, hydroxyl, fluoro or OCH₃.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereineach tetrahydropyran nucleoside analog has Formula Vb and R₄ H. Incertain embodiments, R₄ is hydroxyl. In certain embodiments, R₄ is OCH₃.In certain embodiments, R₄ is fluoro.

In certain embodiments, oligomeric compounds are provided comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vwherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group and whereineach oligomeric compound comprises from about 10 to about 21 monomersubunits in length. In certain embodiments, each oligomeric compoundcomprises from about 10 to about 16 monomer subunits in length. Incertain embodiments, each oligomeric compound comprises from about 10 toabout 14 monomer subunits in length.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula V:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein said oligomeric compound comprises from about 8 to about 40monomeric subunits and is complementary to a target RNA.

In certain embodiments, the cell is in a human. In certain embodiments,the target RNA is selected from mRNA, pre-mRNA and micro RNA. In certainembodiments, the target RNA is mRNA. In certain embodiments, the targetRNA is human mRNA. In certain embodiments, the target RNA is cleavedthereby inhibiting its function.

In certain embodiments, the method further comprises evaluating theantisense activity of the oligomeric compound on said cell. In certainembodiments, the evaluating comprises detecting the levels of targetRNA. In certain embodiments, the evaluating comprises detecting thelevels of a protein. In certain embodiments, the evaluating comprisesdetection of one or more phenotypic effects.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula Vwherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H and R₃ is H. Incertain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is Hand R₄ is OCH₃. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇are each H, R₃ is H and R₄ is fluoro. In certain embodiments, q₁, q₂,q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is H and R₄ is hydroxyl. In certainembodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is H and eachR₄ is H, OCH₃, fluoro or hydroxyl.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula Vwherein each internucleoside linking group is independently aphosphodiester or a phosphorothioate internucleoside linking group. Incertain embodiments, each internucleoside linking group is aphosphorothioate internucleoside linking group.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula Vwherein each tetrahydropyran nucleoside analog of Formula V has theconfiguration shown in Formula Vb:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy; and

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog wherein theoligomeric compound comprises at least one contiguous region of from 1to about 5 tetrahydropyran nucleoside analogs having Formula Va. Incertain embodiments, the oligomeric compound is a blockmer. In certainembodiments, the oligomeric compound is a 3′ or 5′-hemimer.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least two regions of from 1 to about 5 contiguoustetrahydropyran nucleoside analogs that are separated by at least onenucleoside or modified nucleoside. In certain embodiments, theoligomeric compound comprises a gapped oligomeric compound wherein oneexternal region of tetrahydropyran nucleoside analogs is located at the5′-end and a second external region of tetrahydropyran nucleosideanalogs is located at the 3′-end wherein the two external regions areseparated by an internal region comprising from about 6 to about 14monomeric subunits independently selected from nucleosides, modifiednucleosides and tetrahydropyran nucleoside analogs. In certainembodiments, each monomeric subunit in the internal region is aβ-D-2′-deoxyribonucleoside. In certain embodiments, the internal regioncomprises from about 6 to about 14 β-D-2′-deoxyribonucleosides. Incertain embodiments, the internal region comprises from about 10 toabout 12 β-D-2′-deoxyribonucleosides. In certain embodiments, theinternal region comprises from about 10 to about 14β-D-2′-deoxyribonucleosides. In certain embodiments, each externalregion independently comprises from 2 to about 3 tetrahydropyrannucleoside analogs. In certain embodiments, each external regionindependently comprises 2 tetrahydropyran nucleoside analogs. In certainembodiments, each external region independently comprises 2tetrahydropyran nucleoside analogs and the internal region comprises 10β-D-2′-deoxyribonucleosides.

In certain embodiments, methods are provided comprising contacting acell in an animal with an oligomeric compound, said oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula Vwherein each tetrahydropyran nucleoside analog has the Formula andconfiguration shown in Figure Vb:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and

R₄ is H, hydroxyl, fluoro or OCH₃. In certain embodiments, R₄ is H. Incertain embodiments, R₄ is hydroxyl. In certain embodiments, R₄ is OCH₃.In certain embodiments, R₄ is fluoro.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound, said oligomeric compound comprising atleast two contiguous tetrahydropyran nucleoside analogs of Formula V:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁;

said oligomeric compound comprises from about 8 to about 40 monomericsubunits and is complementary to a target RNA; and

wherein at least two of said two contiguous tetrahydropyran nucleosideanalogs are linked by an internucleoside linking group that is otherthan a phosphodiester internucleoside linking group. In certainembodiments, the cell is in an animal. In certain embodiments, the cellis in a human. In certain embodiments, the target RNA is selected frommRNA, pre-mRNA and micro RNA. In certain embodiments, the target RNA ismRNA. In certain embodiments, the target RNA is human mRNA. In certainembodiments, the target RNA is cleaved thereby inhibiting its function.

In certain embodiments, the method further comprises evaluating theantisense activity of said oligomeric compound on said cell. In certainembodiments, the evaluating comprises detecting the levels of targetRNA. In certain embodiments, the evaluating comprises detecting thelevels of a protein. In certain embodiments, the evaluating comprisesdetection of one or more phenotypic effects.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs of Formula V wherein at least two ofthe tetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, R₃ is H. In certain embodiments, R₄ is OCH₃. Incertain embodiments, R₄ is fluoro. In certain embodiments, R₄ ishydroxyl. In certain embodiments, each R₄ is OCH₃, fluoro or hydroxyl.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs of Formula V wherein at least two ofthe tetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein at least two of the tetrahydropyran nucleosideanalogs are linked by a phosphorothioate internucleoside linkage. Incertain embodiments, at least two of said at least two contiguoustetrahydropyran nucleoside analogs are linked by a phosphorus containinginternucleoside linkage other than a phosphodiester internucleosidelinkage. In certain embodiments, at least two of said at least twocontiguous tetrahydropyran nucleoside analogs are linked by a nonphosphorus containing internucleoside linkage. In certain embodiments,at least two of said at least two contiguous tetrahydropyran nucleosideanalogs are linked by a neutral internucleoside linkage. In certainembodiments, each internucleoside linking group is independently aphosphodiester or a phosphorothioate internucleoside linking group. Incertain embodiments, each internucleoside linking group is aphosphorothioate internucleoside linking group.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs wherein at least two of thetetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein each tetrahydropyran nucleoside analog ofFormula V has the configuration shown in Formula Vb:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy; and

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs wherein at least two of thetetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein the oligomeric compound of claim comprises atleast one contiguous region of from 1 to about 5 tetrahydropyrannucleoside analogs having Formula Vb. In certain embodiments, theoligomeric compound comprises a blockmer. In certain embodiments, theoligomeric compound comprises a 3′ or 5′-hemimer.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs wherein at least two of thetetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein the oligomeric compound of claim comprises atleast two contiguous regions of from 1 to about 5 tetrahydropyrannucleoside analogs having Formula Vb wherein the regions are separatedby at least one nucleoside or modified nucleoside. In certainembodiments, the oligomeric compound comprises a gapped oligomericcompound wherein one external region of tetrahydropyran nucleosideanalogs is located at the 5′-end and a second external region oftetrahydropyran nucleoside analogs is located at the 3′-end wherein thetwo external regions are separated by an internal region comprising fromabout 6 to about 14 monomeric subunits independently selected fromnucleosides, modified nucleosides and tetrahydropyran nucleosideanalogs. In certain embodiments, each monomeric subunit in the internalregion is a β-D-2′-deoxyribonucleoside. In certain embodiments, theinternal region comprises from about 6 to about 14β-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises from about 10 to about 12 β-D-2′-deoxyribonucleosides. Incertain embodiments, the internal region comprises from about 10 toabout 14 β-D-2′-deoxyribonucleosides. In certain embodiments, eachexternal region independently comprises from 2 to about 3tetrahydropyran nucleoside analogs. In certain embodiments, eachexternal region independently comprises 2 tetrahydropyran nucleosideanalogs. In certain embodiments, each external region independentlycomprises 2 tetrahydropyran nucleoside analogs and the internal regioncomprises 10 β-D-2′-deoxyribonucleosides.

In certain embodiments, methods are provided comprising contacting acell with an oligomeric compound comprising at least two contiguoustetrahydropyran nucleoside analogs wherein at least two of thetetrahydropyran nucleoside analogs are linked by an internucleosidelinking group that is other than a phosphodiester internucleosidelinking group and wherein each tetrahydropyran nucleoside analog hasFormula V and configuration shown in Formula Vb shown below:

wherein

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and

R₄ is H, hydroxyl, fluoro or OCH₃. In certain embodiments, R₄ is H. Incertain embodiments, R₄ is hydroxyl. In certain embodiments, R₄ is OCH₃.In certain embodiments, R₄ is fluoro.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside analog of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H andR₃ is H In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃is H and R₄ is OCH₃. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ andq₇ are each H, R₃ is H and R₄ is fluoro. In certain embodiments, q₁, q₂,q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is H and R₄ is hydroxyl. In certainembodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H, R₃ is H and eachR₄ is H, OCH₃, fluoro or hydroxyl.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside analog of Formula V wherein each internucleoside linkinggroup is, independently, a phosphodiester or a phosphorothioateinternucleoside linking group. In certain embodiments, eachinternucleoside linking group is a phosphorothioate internucleosidelinking group.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside analog wherein each tetrahydropyran nucleoside analog has theFormula Vb:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy; and

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside comprising at least one contiguous region of from 1 to about5 tetrahydropyran nucleoside analogs and wherein each tetrahydropyrannucleoside analog has Formula Vb. In certain embodiments, the oligomericcompound comprises a blockmer. In certain embodiments, the oligomericcompound comprises a 3′ or 5′-hemimer.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least two regions of from 1 toabout 5 contiguous tetrahydropyran nucleoside analogs that are separatedby at least one nucleoside or modified nucleoside and wherein eachtetrahydropyran nucleoside analog has Formula Vb. In certainembodiments, the oligomeric compound comprises a gapped oligomericcompound wherein one external region of tetrahydropyran nucleosideanalogs is located at the 5′-end and a second external region oftetrahydropyran nucleoside analogs is located at the 3′-end wherein thetwo external regions are separated by an internal region comprising fromabout 6 to about 14 monomeric subunits independently selected fromnucleosides, modified nucleosides and tetrahydropyran nucleosideanalogs. In certain embodiments, essentially each monomeric subunit inthe internal region is a β-D-2′-deoxyribonucleoside. In certainembodiments, the internal region comprises from about 6 to about 14β-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises from about 10 to about 12 β-D-2′-deoxyribonucleosides. Incertain embodiments, the external region independently comprises from 2to about 3 tetrahydropyran nucleoside analogs. In certain embodiments,each external region independently comprises 2 tetrahydropyrannucleoside analogs. In certain embodiments, each external regionindependently comprises 2 tetrahydropyran nucleoside analogs and theinternal region comprises 10 β-D-2′-deoxyribonucleosides.

In certain embodiments, methods of reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside analog wherein each tetrahydropyran nucleoside analog hasFormula Vb:

wherein

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and

R₄ is hydroxyl, fluoro or OCH₃. In certain embodiments, R₄ is hydroxyl.In certain embodiments, R₄ is OCH₃. In certain embodiments, R₄ isfluoro.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving Formula I:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═O—X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, one of R₁ and R₂ is fluoro and the other of R₁and R₂ is H. In certain embodiments, R₁ and R₂ are each fluoro. Incertain embodiments, one of R₁ and R₂ is fluoro and the other of R₁ andR₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments,one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is methyl, ethyl,substituted methyl or substituted ethyl. In certain embodiments, one ofR₁ and R₂ is fluoro and the other of R₁ and R₂ is methyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments,at least one of q₁ and q₂ is methyl. In certain embodiments, at leastone of q₃ and q₄ is methyl. In certain embodiments, at least one of q₅,q₆ and q₇ is methyl.

In certain embodiments, T₁ and T₂ are each H. In certain embodiments, atleast one of T₁ and T₂ is a hydroxyl protecting group. In certainembodiments, each hydroxyl protecting group is, independently, acetyl,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl,2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4-methoxytrityl, 4,4′-dimethoxytrityl, trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl,trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triflate, trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl or substituted pixyl. In certainembodiments, T₁ is acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, 4-methoxytrityl or 4,4′-dimethoxytrityl. Incertain embodiments, one of T₁ and T₂ is a hydroxyl protecting group andthe other of T₁ and T₂ is diisopropylcyanoethoxy phosphoramidite orH-phosphonate. In certain embodiments, T₁ is 4,4′-dimethoxytrityl and T₂is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, Bx is uracil, thymine, cytosine, adenine orguanine. In certain embodiments, Bx is a pyrimidine, substitutedpyrimidine, purine or substituted purine wherein said substitution isother than an intercalator or a linked group that does not interact witha nucleic acid target when the tetrahydropyran nucleoside analog islocated in an oligomeric compound. In certain embodiments, Bx is uracil,5-methyluracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil,thymine, 2′-thio-thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine,2,6-diaminopurine, 1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certainembodiments, Bx is uracil, 5-methyluracil, 5-propynyl-uracil, thymine,cytosine, 5-methylcytosine, 5-propynyl-cytosine, adenine or guanine.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving the configuration shown in Formula Ia:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, a tetrahydropyran nucleoside analog is providedhaving the configuration shown in Formula Ia wherein R₂ is fluoro. Incertain embodiments, a tetrahydropyran nucleoside analog is providedhaving the configuration shown in Formula Ia wherein R₁ is H and R₂ isfluoro. In certain embodiments, a tetrahydropyran nucleoside analog isprovided having the configuration shown in Formula Ia wherein R₁ is H,R₂ is fluoro and q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H.

In certain embodiments, a tetrahydropyran nucleoside analog is providedhaving the configuration shown in Formula Ia wherein R₁ is C₁-C₆ alkylor substituted C₁-C₆ alkyl and R₂ is fluoro. In certain embodiments, atetrahydropyran nucleoside analog is provided having the configurationshown in Formula Ia wherein R₁ is methyl, ethyl, substituted methyl orsubstituted ethyl and R₂ is fluoro.

In certain embodiments, a tetrahydropyran nucleoside analog is providedhaving the configuration shown in Formula Ia wherein R₁ and R₂ are eachfluoro.

In certain embodiments, oligomeric compounds each comprising at leastone tetrahydropyran nucleoside analog of Formula II are provided:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a protecting group, a linked conjugate group ora 5′ or 3′-terminal group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs.

In certain embodiments, one of R₁ and R₂ is fluoro and the other of R₁and R₂ is H. In certain embodiments, R₁ and R₂ are each fluoro. Incertain embodiments, one of R₁ and R₂ is fluoro and the other of R₁ andR₂ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments,one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is methyl, ethyl,substituted methyl or substituted ethyl. In certain embodiments, one ofR₁ and R₂ is fluoro and the other of R₁ and R₂ is methyl.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certain embodiments, at leastone of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments,at least one of q₁ and q₂ is methyl. In certain embodiments, at leastone of q₃ and q₄ is methyl. In certain embodiments, at least one of q₅,q₆ and q₇ is methyl.

In certain embodiments, at least one of T₃ and T₄ is a linked conjugategroup.

In certain embodiments, each internucleoside linking group is,independently, a phosphodiester or a phosphorothioate. In certainembodiments, each internucleoside linking group is a phosphorothioate.

In certain embodiments, Bx is uracil, thymine, cytosine, adenine orguanine. In certain embodiments, Bx is a pyrimidine, substitutedpyrimidine, purine or substituted purine wherein said substitution isother than an intercalator or a linked group that does not interact witha nucleic acid target when the tetrahydropyran nucleoside analog islocated in an oligomeric compound. In certain embodiments, Bx is uracil,5-methyluracil, 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil,thymine, 2′-thio-thymine, cytosine, 5-methylcytosine,5-thiazolo-cytosine, 5-propynyl-cytosine, adenine, guanine,2,6-diaminopurine, 1H-pyrimido[5,4-b][1,4benzoxazin-2(3H)-one),1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one,2H-pyrimido[4,5-b]indol-2-one orH-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one. In certainembodiments, Bx is uracil, 5-methyluracil, 5-propynyl-uracil, thymine,cytosine, 5-methylcytosine, 5-propynyl-cytosine, adenine or guanine.

In certain embodiments, oligomeric compounds are provided comprising atleast one tetrahydropyran nucleoside analog having the configurationshown in Formula IIa:

wherein

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a protecting group, a linked conjugate group ora 5′ or 3′-terminal group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs.

In certain embodiments, oligomeric compounds are provided comprising atleast one tetrahydropyran nucleoside analog having the configurationshown in Formula IIa wherein R₂ is fluoro. In certain embodiments,oligomeric compounds are provided comprising at least onetetrahydropyran nucleoside analog having the configuration shown inFormula IIa wherein R₁ is H and R₂ is fluoro. In certain embodiments,oligomeric compounds are provided comprising at least onetetrahydropyran nucleoside analog having the configuration shown inFormula IIa wherein R₁ is H, R₂ is fluoro and q₁, q₂, q₃, q₄, q₅, q₆ andq₇ are each H.

In certain embodiments, oligomeric compounds are provided comprising atleast one contiguous region of from 1 to about 5 tetrahydropyrannucleoside analogs, each having the configuration shown in Formula IIa.In certain embodiments, oligomeric compounds are provided comprising ablockmer motif having at least one contiguous region of from 1 to about5 tetrahydropyran nucleoside analogs, each having the configurationshown in Formula IIa. In certain embodiments, oligomeric compounds areprovided comprising a 3′ or 5′-hemimer motif having at least onecontiguous region of from 1 to about 5 tetrahydropyran nucleosideanalogs, each having the configuration shown in Formula IIa.

In certain embodiments, oligomeric compounds are provided comprising atleast one contiguous region of from 1 to about 5 tetrahydropyrannucleoside analogs, each having the Formula and configuration:

In certain embodiments, oligomeric compounds are provided comprising atleast two regions of from 1 to about 5 contiguous tetrahydropyrannucleoside analogs, each having Formula II, that are separated by atleast one nucleoside or modified nucleoside. In certain embodiments,oligomeric compounds are provided comprising a gapped motif having atleast two regions of from 1 to about 5 contiguous tetrahydropyrannucleoside analogs, each having Formula II, wherein one region oftetrahydropyran nucleoside analogs is located at the 5′-end and theother region of tetrahydropyran nucleoside analogs is located at the3′-end and wherein the two regions of tetrahydropyran nucleoside analogsare separated by an internal region comprising from about 6 to about 14monomeric subunits independently selected from nucleosides, modifiednucleosides and tetrahydropyran nucleoside analogs. In certainembodiments, each monomeric subunit in the internal region is aβ-D-2′-deoxyribonucleoside. In certain embodiments, the internal regioncomprises from about 6 to about 14 β-D-2′-deoxyribonucleosides. Incertain embodiments, the internal region comprises from about 10 toabout 12 β-D-2′-deoxyribonucleosides. In certain embodiments, eachregion of tetrahydropyran nucleoside analogs independently comprisesfrom 2 to about 3 tetrahydropyran nucleoside analogs. In certainembodiments, each region of tetrahydropyran nucleoside analogsindependently comprises 2 tetrahydropyran nucleoside analogs. In certainembodiments, the internal region comprises 10β-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises 10 β-D-2′-deoxyribonucleosides and each tetrahydropyrannucleoside analog has the Formula and configuration:

In certain embodiments, oligomeric compounds are provided comprising agapped motif having at least two regions of from 1 to about 5 contiguoustetrahydropyran nucleoside analogs, each having Formula II, wherein oneregion of tetrahydropyran nucleoside analogs is located at the 5′-endand the other region of tetrahydropyran nucleoside analogs is located atthe 3′-end, the two regions of tetrahydropyran nucleoside analogs areseparated by an internal region comprising from about 6 to about 14monomeric subunits independently selected from nucleosides, modifiednucleosides and tetrahydropyran nucleoside analogs and the oligomericcompounds further comprise a 3′-terminal group. In certain embodiments,the 3′-terminal group comprises from 1 to about 4 modified or unmodifiednucleosides.

In certain embodiments oligomeric compounds are provided wherein eacholigomeric compound includes at least one tetrahydropyran nucleosideanalog of Formula II comprising from about 10 to about 21 nucleosidesand or nucleoside analogs in length. In certain embodiments, eacholigomeric compound including at least one tetrahydropyran nucleosideanalog of Formula II comprises from about 10 to about 16 nucleosides andor nucleoside analogs in length. In certain embodiments, each oligomericcompound including at least one tetrahydropyran nucleoside analog ofFormula II comprises from about 10 to about 14 nucleosides and ornucleoside analogs in length.

In certain embodiments, methods for reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound oligomeric compound including at least onetetrahydropyran nucleoside analog of Formula II.

In certain embodiments, methods for reducing target messenger RNA areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound comprising at least one tetrahydropyrannucleoside analog of Formula III:

Wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a protecting group, a linked conjugate group ora 5′ or 3′-terminal group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

each R₃ and R₄ is, independently, H, hydroxyl, fluoro, C₁-C₆ alkoxy,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs.

In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H and R₃ isH. In certain embodiments, q₁, q₂, q₃, q₄, q₅, C₆ and q₇ are each H, R₃is H and R₄ is OCH₃. In certain embodiments, q₁, q₂, q₃, q₄, q₅, q₆ andq₇ are each H, R₃ is H and R₄ is fluoro. In certain embodiments, q₁, q₂,q₃, q₄, q₅, C₁₆ and q₇ are each H, R₃ is H and R₄ is hydroxyl.

In certain embodiments, each tetrahydropyran nucleoside analog in eachof the oligomeric compounds used in the methods has the Formula andconfiguration:

Wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a protecting group, a linked conjugate group ora 5′ or 3′-terminal group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

each R₃ and R₄ is, independently, H, hydroxyl, fluoro, C₁-C₆ alkoxy,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain embodiments, the oligomeric compounds used in the methodscomprise a gapped motif wherein one external region of tetrahydropyrannucleoside analogs is located at the 5′-end and a second external regionof tetrahydropyran nucleoside analogs is located at the 3′-end whereinthe two external regions are separated by an internal region comprisingfrom about 6 to about 14 monomeric subunits independently selected fromnucleosides, modified nucleosides and tetrahydropyran nucleosideanalogs.

In certain embodiments, essentially each monomeric subunit in theinternal region is a β-D-2′-deoxyribonucleoside. In certain embodiments,the internal region comprises from about 6 to about 14β-D-2′-deoxyribonucleosides. In certain embodiments, the internal regioncomprises from about 10 to about 12 β-D-2′-deoxyribonucleosides. Incertain embodiments, each external region independently comprises from 2to about 3 tetrahydropyran nucleoside analogs. In certain embodiments,each external region independently comprises 2 tetrahydropyrannucleoside analogs. In certain embodiments, the internal regioncomprises 10 β-D-2′-deoxyribonucleosides.

In certain embodiments, each tetrahydropyran nucleoside analog used inthe present methods has the Formula and configuration:

Wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a protecting group, a linked conjugate group ora 5′ or 3′-terminal group; and

R₄ is hydroxyl, fluoro or OCH₃.

In certain embodiments, R₄ is hydroxyl. In certain embodiments, R₄ isOCH₃. In certain embodiments, R₄ is fluoro.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are tetrahydropyran nucleoside analogs, oligomericcompounds that include such analogs and methods of using the oligomericcompounds. Also included are intermediates and methods for preparing thetetrahydropyran nucleoside analogs and the oligomeric compounds. Thetetrahydropyran nucleoside analogs each have a core structure comprisinga tetrahydropyran ring. Attached to one of the two carbon atoms flankingthe oxygen atom is a first group capable of forming an internucleosidelinkage and attached to the carbon atom next to the other flankingcarbon atom (one carbon removed from the oxygen atom) is a heterocyclicbase moiety. The heterocyclic base moiety can be optionally substitutedwith groups to enhance the affinity for a complementary base in a secondstrand such as a nucleic acid target. In certain embodiments, thetetrahydropyran nucleoside analogs further comprise at least onefluorine atom adjacent to the heterocyclic base on the carbon furthestfrom the ring oxygen atom. The carbon atom having the fluorine atom canbe further substituted or not.

In certain embodiments, the tetrahydropyran nucleoside analogs haveFormula XVI:

wherein: Bx is a heterocyclic base moiety; T₅ is a hydroxyl protectinggroup; L₁ is H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; Z₁ isO⁻ or OE₁; Z₂ is OH, OE₁ or N(E₁)(E₂); each E₁ and E₂ is, independently,alkyl or substituted alkyl; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each,independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, the tetrahydropyran nucleoside analogs have theconfiguration of Formula XVII:

In certain embodiments, the tetrahydropyran nucleoside analog of FormulaXVII is further defined wherein: q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are eachH; Bx is uracil, 5-methyluracil, thymine, cytosine, 5-methylcytosine,2,6-diaminopurine, adenine or guanine; T₅ is 4,4′-dimethoxytrityl; Z₁ isO(CH₂)₂CN; and Z₂ is N[CH₂(CH₃)₂]₂.

In certain embodiments, the oligomeric compounds provided hereincomprise at least one tetrahydropyran nucleoside analog of Formula X:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X: Bx is a heterocyclic base moiety; T₃ andT₄ are each, independently, an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound or one ofT₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ areeach independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl; one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H,halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl; each substituted groupcomprises one or more optionally protected substituent groupsindependently selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁,J₂ and J₃ is, independently, H or C₁-C₆ alkyl; and wherein saidoligomeric compound comprises from about 8 to about 40 monomer subunitslinked by internucleoside linking groups and at least oneinternucleoside linking group is a phosphorothioate internucleosidelinking group.

In certain embodiments, each of the oligomeric compounds provided hereincomprise at least one tetrahydropyran nucleoside analog of Formula XI:

In certain embodiments, each of the tetrahydropyran nucleoside analogsin each of the oligomeric compounds provided herein has Formula XII:

In certain embodiments, the oligomeric compound provided herein compriseat least two tetrahydropyran nucleoside analogs of Formula XIII:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula XIII: Bx is a heterocyclic base moiety; T₃ and T₄ areeach, independently, an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound or one ofT₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ areeach independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl; R₃ and R₄ are each independently, H, hydroxyl, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆alkoxy; each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl; wherein said oligomeric compound comprises from about 8 to about40 monomer subunits; and at least two of the tetrahydropyran nucleosideanalogs of Formula XIII are linked by a phosphorothioate internucleosidelinking group.

In certain embodiments, the oligomeric compounds provided hereincomprise at least two tetrahydropyran nucleoside analogs of Formula XIIIwherein each tetrahydropyran nucleoside analog also has theconfiguration of Formula XIV:

In certain embodiments, the oligomeric compounds provided hereincomprise at least two tetrahydropyran nucleoside analogs of Formula XIIIwherein at least one tetrahydropyran nucleoside analog has Formula XV:

wherein: Bx is a heterocyclic base moiety; and R₅ is H, OCH₃ or F.

In certain embodiments, methods comprising contacting a cell in ananimal with one or more of the oligomeric compounds disclosed herein areprovided. In certain embodiments, the cell is in a human.

In certain embodiments, tetrahydropyran nucleoside analogs are providedhaving Formula I:

wherein:

Bx is a heterocyclic base moiety;

one of T₁ and T₂ is H or a hydroxyl protecting group and the other of T₁and T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

each q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and

wherein each substituted group comprises one or more optionallyprotected substituent groups independently selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S orNJ₁.

In certain embodiments, tetrahydropyran nucleosides are provided havingthe configuration shown in Formula Ia:

Wherein the configuration has been defined but the variables are definedthe same as for Formula I above.

In certain embodiments, tetrahydropyran nucleosides are provided havingFormula II:

Wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds are provided comprising atleast one tetrahydropyran nucleoside analog of Formula III:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula III:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

one of R₁ and R₂ is fluoro and the other of R₁ and R₂ is H, halogen,C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein said oligomeric compound comprises from about 8 to about 40monomeric subunits.

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog of Formula III has the configurationshown below in Formula IIIa:

Wherein the configuration has been defined but the variables are definedthe same as for Formula III above.

In certain embodiments, oligomeric compounds are provided wherein eachtetrahydropyran nucleoside analog has Formula IV:

Wherein:

Bx is a heterocyclic base moiety.

In certain embodiments, oligomeric compounds are provided having atleast two contiguous tetrahydropyran nucleoside analogs of Formula V:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁;

said oligomeric compound comprises from about 8 to about 40 monomericsubunits; and

wherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group.

In certain embodiments, oligomeric compounds are provided having atleast two contiguous tetrahydropyran nucleoside analogs of Formula Va:

Wherein the configuration has been defined but the variables are definedthe same as for Formula V above and wherein each oligomeric compoundcomprises from about 8 to about 40 monomeric subunits; and

wherein for each oligomeric compound at least two of the tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group.

In certain embodiments, oligomeric compounds are provided having atleast two contiguous tetrahydropyran nucleoside analogs of Formula Vb:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and

R₄ is H, hydroxyl, fluoro or OCH₃.

In certain embodiments, methods of using the oligomeric compounds areprovided comprising contacting a cell in an animal with an oligomericcompound, said oligomeric compound comprising at least onetetrahydropyran nucleoside analog of Formula V:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₉ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein said oligomeric compound comprises from about 8 to about 40monomeric subunits and is complementary to a target RNA.

In one aspect, methods are provided comprising contacting a cell with anoligomeric compound, said oligomeric compound comprising at least twocontiguous tetrahydropyran nucleoside analogs of Formula V:

wherein independently for each of said tetrahydropyran nucleosideanalogs of Formula V:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁;

said oligomeric compound comprises from about 8 to about 40 monomericsubunits and is complementary to a target RNA; and

wherein at least two of said at least two contiguous tetrahydropyrannucleoside analogs are linked by an internucleoside linking group thatis other than a phosphodiester internucleoside linking group.

In certain embodiments, methods are provided comprising contacting oneor more cells, a tissue or an animal with an oligomeric compoundcomprising at least one tetrahydropyran nucleoside analog of Formula V:

Wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl;

R₃ and R₄ are each independently, H, hydroxyl, fluoro, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, NJ₁J₂, SJ₁,N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ andJ₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

wherein the oligomeric compound comprises from about 8 to about 40nucleosides, modified nucleosides and or tetrahydropyran nucleosideanalogs.

In certain embodiments, the methods are performed when the cell is in ahuman and the target RNA is a mRNA.

The groups capable of forming internucleoside linkages can be variable.In certain embodiments, groups capable of forming internucleosidelinkages include optionally protected primary and secondary alcohols andreactive phosphorus groups. In certain embodiments, one of the groupscapable of forming an internucleoside linkage is an optionally protectedhydroxymethylene and the other group is an optionally protected hydroxylor reactive phosphorus group.

Two different tetrahydropyran nucleoside analogs were incorporated intothe wings of 2/10/2 gapped oligomeric compounds and compared to a 2/10/2gapped oligomeric compound having 2′-O-MOE modified nucleosides in thewings. In each of the oligomeric compounds the 10 nucleosides in the gapare each a β-D-2′-deoxyribonucleoside, the wings are uniformly modifiedand each internucleoside linkage is a phosphorothioate. The gappedoligomeric compounds were evaluated for their ability to inhibit PTENboth in vitro and in vivo. The Formula and configuration of thetetrahydropyran nucleoside analogs and the 2′-O-MOE modified nucleosideis shown below:

The oligomeric compounds having 3′-O—CH₃ and 3′-F tetrahydropyrannucleoside analogs demonstrated enhanced in vitro and in vivo activitycompared to 2′-O-MOE modified nucleosides with the 3′-F demonstratingthe highest level of reduction compared to the untreated control (seeexamples 31 and 33). The enhanced in vitro activity of oligomericcompounds incorporating either the 3′-O—CH₃ or the 3′-F tetrahydropyrannucleoside analogs was not predicted by the binding affinities of themodifications (Tm: 2′-O-MOE>3′-F>3′-O—CH₃). Oligomeric compounds havingthe 3′-O—CH₃ or 3′-F tetrahydropyran nucleoside analogs each have alower Tm than that for the oligomeric compound having 2′-O-MOE modifiednucleosides.

This level of activity is also unexpected based on previous published invitro data. According to Published US Patent Application US 2004/0033967the Tm of an oligomeric compound having uniform 3′-H tetrahydropyrannucleoside analogs was determined against RNA. Each 3′-O—CH₃tetrahydropyran nucleoside analog incorporated into the uniform 3′-Holigomeric compound increased the Tm for RNA by only 0.4° C. permodification.

It has been previously reported (Kang et al., Nucleic Acids Research,2004, 32(14), 4411-4419) that the activity of a gapped oligomericcompound having phosphodiester linked 3′-H tetrahydropyran nucleosideanalogs in the wings and phosphorothioate linkedβ-D-2′-deoxyribonucleosides in the gap was compared to that of a similargapped oligomeric compound having full phosphorothioate internucleosidelinkages and 2′-O-MOE modified nucleosides in the wings. It was reportedthat the gapmer having 3′-H tetrahydropyran nucleoside analogs showed invitro activity that was similar to the MOE gapmer. It was furtherreported that the gapmer having 3′-H tetrahydropyran nucleoside analogsshowed toxicity at higher concentrations (Kang, ibid). Kang et al.,suggested that removing the phosphorothioate internucleoside linkagesfrom the deoxyribonucleotide gap segment might reduce the observedcytotoxicity while maintaining the required nuclease resistance andtarget binding.

The in vitro data reported herein for gapped oligomeric compounds (fullphosphorothioate linked gapmers) having β-D-2′-deoxyribonucleosides inthe gap and either 3′-OCH₃ or 3′-F tetrahydropyran nucleoside analogs inthe wings showed a modest increase in activity over the gapmers having2′-O-MOE nucleosides in the wings. The 2′-O-MOE gapmer had an IC₅₀ of 37compared to IC₅₀'s of 23 and 16 for the gapmers having 3′-O—CH and 3′-Ftetrahydropyran nucleoside analogs respectively. The lower IC₅₀ for eachof the tetrahydropyran nucleoside analogs relative to the 2′-O-MOEoligomer is unexpected because the structures and the Tm data for eachof these tetrahydropyran nucleoside analogs are similar to the 3-Hnucleoside analog reported in Kang.

In addition to possessing increased in vitro activity as compared to the2′-O-MOE gapmer, the gapmers having either 3′-F or 3′-OCH₃tetrahydropyran nucleoside analogs in the wings andβ-D-2′-deoxyribonucleosides in the gap exhibited in vivo potency thatwas, for the higher dose in the study, not predicted by the Tm or the invitro activity of the compounds. Compared to the 2′-β-MOE gapmer the3′-O—CH₃ gapmer showed a two fold increase in potency and the 3′-Fgapmer showed an eight fold increase in potency.

Also unexpected was the level of in vitro and in vivo activity of gappedoligomeric compounds having 3′-F tetrahydropyran nucleoside analogscompared to gapped oligomeric compounds having locked nucleosides havinga 4′-CH₂—O-2′ bridged sugars. The gapped oligomeric compounds havingthese motifs (examples 32 and 35) exhibit very high levels of in vitroand in vivo activity and in each study the levels between the twochemistries is essentially equal. The Tm to complementary RNA as shownherein is 60.5° C. for the gapped oligomeric compound having the lockednucleosides and 52.6 (50.7 w/out 5′-CH₃ groups on the monomers in thewings 2/10/2 motif) for the gapped oligomeric compound having the 3′-Ftetrahydropyran modified nucleoside analogs. This is an 8-10° C.difference for the oligomeric compound with the locked nucleosideshaving 4′-CH₂—O-2′ bridged sugars. The level of in vitro and in vivoactivity of oligomeric compounds having 3′-F tetrahydropyran nucleosideanalogs and locked nucleosides having 4′-CH₂—O-2′ bridged sugars in thewings is unexpected based on the 8-10° C. difference in Tm.

In addition to enhanced activity the tetrahydropyran nucleoside analogsalso exhibit lower toxicity when compared to a locked nucleoside asevidenced in the in vivo examples. The ALT and AST levels are extremelyelevated in the high dose group for the locked nucleosides having the4′-CH₂—O-2′ bridge (Example 35). The ALT and ASTs for the differentgapped oligomeric compounds (2/10/2 and 2/14/2 motifs) having theselected tetrahydropyran nucleoside analog do not show a significantincrease.

In addition to having enhanced activity the tetrahydropyran nucleosideanalogs are also expected to be useful for enhancing desired propertiesof oligomeric compounds in which they are incorporated such as nucleaseresistance. Oligomeric compounds comprising such tetrahydropyrannucleoside analogs are also expected to be useful as primers and probesin various diagnostic applications.

In certain embodiments, tetrahydropyran nucleoside analogs are usefulfor modifying oligomeric compounds at one or more positions. Suchmodified oligomeric compounds can be described as having a particularmotif. In certain embodiments, the motifs include without limitation, agapped motif, a hemimer motif, a blockmer motif, a fully modified motif,a positionally modified motif and an alternating motif. In conjunctionwith these motifs a wide variety of linkages can also be used includingbut not limited to phosphodiester and phosphorothioate linkages useduniformly or in combinations. The positioning of tetrahydropyrannucleoside analogs and the use of linkage strategies can be easilyoptimized to enhance activity for a selected target. Such motifs can befurther modified by the inclusion of a 5′ or 3′-terminal group such as aconjugate group.

The term “motif” refers to the pattern of nucleosides in an oligomericcompound. The pattern is dictated by the positioning of nucleosideshaving unmodified (β-D-ribonucleosides and/orβ-D-2′-deoxyribonucleosides) and/or modified sugar groups within anoligomeric compound. The type of heterocyclic base and internucleosidelinkages used at each position is variable and is not a factor indetermining the motif of an oligomeric compound. The presence of one ormore other groups including but not limited to capping groups andconjugate groups is also not a factor in determining the motif.

Representative U.S. patents that teach the preparation of representativemotifs include, but are not limited to, U.S. Pat. Nos. 5,013,830;5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety. Motifs are alsodisclosed in International Applications PCT/US2005/019219, filed Jun. 2,2005 and published as WO 2005/121371 on Dec. 22, 2005 andPCT/US2005/019220, filed Jun. 2, 2005 and published as WO 2005/121372 onDec. 22, 2005; each of which is incorporated by reference herein in itsentirety.

As used herein the term “alternating motif” is meant to include acontiguous sequence of nucleosides comprising two different monomersubunits that alternate for essentially the entire sequence of theoligomeric compound. The pattern of alternation can be described by theformula: 5′-A(-L-B-L-A)_(n)(-L-B)_(nn)-3′ where one of each A or each Bis a tetrahydropyran nucleoside analog and the other of each A or B is amonomer subunit that is other than a tetrahydropyran nucleoside, each Lis an internucleoside linking group, nn is 0 or 1 and n is from about 4to about 12. This permits alternating oligomeric compounds from about 9to about 26 monomer subunits in length. This length range is not meantto be limiting as longer and shorter oligomeric compounds are alsoamenable to the present invention. This formula also allows for even andodd lengths for alternating oligomeric compounds.

In certain embodiments, the other of each A or B is selected fromβ-D-ribonucleosides, 2′-modified nucleosides, 4′-thio modifiednucleosides, 4′-thio-2′-modified nucleosides, bicyclic sugar modifiednucleosides and other modified nucleosides. The alternating motif is notdefined by the nucleobase sequence or the internucleoside linkages.

As used herein the term “fully modified motif” is meant to include acontiguous sequence of monomer subunits that have the same sugar orsugar surrogate group. In certain embodiments, the fully modified motifincludes a contiguous sequence of tetrahydropyran nucleoside analogs. Incertain embodiments, the 3′ and 5′-terminal ends comprise unmodifiednucleosides.

As used herein the term “hemimer motif” is meant to include anoligomeric compound having contiguous sequence of monomer subunits ofone type with a contiguous sequence of monomer subunits of a second typelocated at one of the termini. The two types of monomer subunits aredifferentiated by the type of sugar or sugar surrogate group comprisingthe nucleosides and is independent of the type of base and linkage used.Sugar surrogate groups includes other than ribose type sugars such asthe presently described tetrahydropyran nucleoside analogs wherein atetrahydropyran ring is used in place of the ribose ring. In certainembodiments, the sugar surrogate group is a tetrahydropyran moietycomprising a tetrahydropyran nucleoside analog. In certain embodiments,the hemimer motif comprises a contiguous sequence of from about 10 toabout 28 monomer subunits of one type with from 1 to 5 or from 2 to 5monomer subunits of a second type located at one of the termini. Incertain embodiments the hemimer is a contiguous sequence of from about 8to about 20 β-D-2′-deoxyribonucleosides having from 1-12 contiguoustetrahydropyran nucleoside analogs located at one of the termini. Incertain embodiments, In certain embodiments the hemimer is a contiguoussequence of from about 8 to about 20 β-D-2′-deoxyribonucleosides havingfrom 1-5 contiguous tetrahydropyran nucleoside analogs located at one ofthe termini. In certain embodiments the hemimer is a contiguous sequenceof from about 12 to about 18 β-D-2′-deoxyribonucleosides having from 1-3contiguous tetrahydropyran nucleoside analogs located at one of thetermini.

As used herein the term “blockmer motif” is meant to include anoligomeric compound having a contiguous sequence of monomer subunits ofone type with a contiguous sequence of monomer subunits of a second typelocated at internally. The two types of monomer subunits aredifferentiated by the type of sugar or sugar surrogate group comprisingthe nucleosides and is independent of the type of base and linkage used.A blockmer overlaps somewhat with a gapmer in the definition buttypically only the monomer subunits in the block are modified in ablockmer and only the monomer subunits in the external regions aremodified in a gapmer. In certain embodiments, blockmers can have othertypes of modified monomer subunits throughout the oligomeric compound atpositions not occupied by the block.

As used herein the term “positionally modified motif” is meant toinclude a sequence of monomer subunits of one type that is interruptedwith two or more regions of from 1 to about 5 modified monomer subunitsmonomer subunits of one type. In certain embodiments, a positionallymodified oligomeric compound is a sequence of from 8 to 20β-D-2′-deoxyribonucleosides that further includes two or three regionsof from 2 to about 5 contiguous tetrahydropyran nucleoside each.Positionally modified oligomeric compounds are distinguished from gappedmotifs, hemimer motifs, blockmer motifs and alternating motifs becausethe pattern of regional substitution defined by any positional motif isnot defined by these other motifs. Positionally modified motifs are notdetermined by the nucleobase sequence or the location or types ofinternucleoside linkages. The term positionally modified oligomericcompound includes many different specific substitution patterns.

As used herein the term “gapmer” or “gapped oligomeric compound” ismeant to include a contiguous sequence of nucleosides that is dividedinto 3 regions, an internal region having an external region on each ofthe 5′ and 3′ ends. The regions are differentiated from each other atleast by having different sugar or sugar surrogate groups that comprisethe nucleosides. In certain embodiments, the external regions are each,independently, from 1 to about 5 modified nucleosides and the internalregion is from 6 to 18 nucleosides. The types of nucleosides that aregenerally used to differentiate the regions of a gapped oligomericcompound include, but are not limited to, β-D-ribonucleosides,β-D-2′-deoxyribonucleosides, 2′-modified nucleosides, 4′-thio modifiednucleosides, 4′-thio-2′-modified nucleosides, bicyclic sugar modifiednucleosides and sugar surrogate containing nucleosides such astetrahydropyran nucleoside analogs. Each of the regions of a gappedoligomeric compound is essentially uniformly modified e.g. the sugar orsugar surrogate groups are identical with at least the internal regionhaving different sugar groups than each of the external regions. Theinternal region or the gap generally comprisesβ-D-2′-deoxyribonucleosides but can be a sequence of sugar modifiednucleosides.

In certain embodiments, the gapped oligomeric compounds comprise aninternal region of β-D-2′-deoxyribonucleosides with one of the externalregions comprising tetrahydropyran nucleoside analogs as disclosedherein. In certain embodiments, the gapped oligomeric compounds comprisean internal region of β-D-2′-deoxyribonucleosides with both of theexternal regions comprising tetrahydropyran nucleoside analogs asdisclosed herein. In certain embodiments, the gapped oligomericcompounds comprise an internal region of β-D-2′-deoxyribonucleosideswith both of the external regions comprising tetrahydropyran nucleosideanalogs having Formula II. A further example of a gapped motif is shownin Example 32 and 35 where an oligomeric compound comprising 14nucleosides has 2 bicyclic nucleosides positioned at each of the 3′ and5′ ends and further includes 10 unmodified β-D-2′-deoxyribonucleosidesin the internal region. This oligomeric compound has a gapped motifwherein the terminal externa regions of bicyclic nucleosides areconsidered the wings and the β-D-2′-deoxyribonucleoside internal regionis considered the gap.

In certain embodiments, gapped oligomeric compounds are providedcomprising one or two tetrahydropyran nucleoside analogs at the 5′-end,two or three tetrahydropyran nucleoside analogs at the 3′-end and aninternal region of from 10 to 16 nucleosides. In certain embodiments,gapped oligomeric compounds are provided comprising one tetrahydropyrannucleoside analog at the 5′-end, two tetrahydropyran nucleoside analogsat the 3′-end and an internal region of from 10 to 16 nucleosides. Incertain embodiments, gapped oligomeric compounds are provided comprisingone tetrahydropyran nucleoside analog at the 5′-end, two tetrahydropyrannucleoside analogs at the 3′-end and an internal region of from 10 to 14nucleosides. In certain embodiments, the internal region is essentiallya contiguous sequence of β-D-2′-deoxyribonucleosides. In anotherembodiment, oligomeric compounds are provided that further include, butare not limited to, one or more 5′ or 3′-terminal groups such as furthermodified or unmodified nucleosides, linked conjugate groups and othergroups known to the art skilled.

In certain embodiments, gapped oligomeric compounds are provided thatare from about 10 to about 21 nucleosides in length. In anotherembodiment, gapped oligomeric compounds are provided that are from about12 to about 16 nucleosides in length. In a further embodiment, gappedoligomeric compounds are provided that are from about 12 to about 14nucleosides in length.

In one aspect, oligomeric compounds are provided comprisingtetrahydropyran nucleoside analogs having formula III. In anotheraspect, oligomeric compounds are provided comprising tetrahydropyrannucleoside analogs having formula IIIa. In another aspect, oligomericcompounds are provided comprising tetrahydropyran nucleoside analogshaving formula IV. In another aspect, oligomeric compounds are providedcomprising tetrahydropyran nucleoside analogs having formula V. Inanother aspect, oligomeric compounds are provided comprisingtetrahydropyran nucleoside analogs having formula Va. In another aspect,oligomeric compounds are provided comprising tetrahydropyran nucleosideanalogs having formula Vb.

The terms “substituent” and “substituent group,” as used herein, aremeant to include groups that are typically added to other groups orparent compounds to enhance desired properties or give desired effects.Substituent groups can be protected or unprotected and can be added toone available site or to many available sites in a parent compound.Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound. Such groupsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic, heteroaryl, heteroarylalkyl, amino (—NR_(bb)R_(cc)),imino(═NR_(bb)), amido (—C(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)R_(aa)),azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)NR_(bb)R_(cc)or —N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)NR_(bb)R_(cc)),thioureido (—N(R_(bb))C(S)NR_(bb)R_(cc)), guanidinyl(—N(R_(bb))C(═NR_(bb))NR_(bb)R_(cc)), amidinyl(—C(═NR_(bb))NR_(bb)R_(cc) or —N(R_(bb))C(NR_(bb))R_(aa)), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)),sulfonamidyl (—S(O)₂NR_(bb)R_(cc) or —N(R_(bb))S(O)₂R_(bb)) andconjugate groups. Wherein each R_(aa), R_(bb) and R_(cc) is,independently, H, an optionally linked chemical functional group or afurther substituent group with a preferred list including, withoutlimitation H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl,aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.Selected substituents within the compounds described herein are presentto a recursive degree.

In this context, “recursive substituent” means that a substituent mayrecite another instance of itself. Because of the recursive nature ofsuch substituents, theoretically, a large number may be present in anygiven claim. One of ordinary skill in the art of medicinal chemistry andorganic chemistry understands that the total number of such substituentsis reasonably limited by the desired properties of the compoundintended. Such properties include, by way of example and not limitation,physical properties such as molecular weight, solubility or log P,application properties such as activity against the intended target, andpractical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in a claim of the invention, the total numberwill be determined as set forth above.

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred. The term “lower alkyl” asused herein includes from 1 to about 6 carbon atoms. Alkyl groups asused herein may optionally include one or more further substitutentgroups.

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atoms andhaving at least one carbon-carbon double bond. Examples of alkenylgroups include, but are not limited to, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substitutent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substitutent groups.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general Formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substitutent groups.

The term “alicyclic” or “alicyclyl” refers to a cyclic ring systemwherein the ring is aliphatic. The ring system can comprise one or morerings wherein at least one ring is aliphatic. Preferred alicyclicsinclude rings having from about 5 to about 9 carbon atoms in the ring.Alicyclic as used herein may optionally include further substitutentgroups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitationpolyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstitutent groups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy andthe like. Alkoxy groups as used herein may optionally include furthersubstitutent groups.

The term “aminoalkyl” as used herein, refers to an amino substitutedalkyl radical. This term is meant to include C₁-C₁₂ alkyl groups havingan amino substituent at any position and wherein the alkyl groupattaches the aminoalkyl group to the parent molecule. The alkyl and/oramino portions of the aminoalkyl group can be further substituted withsubstituent groups.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to a radicalformed between an alkyl group and an aryl group wherein the alkyl groupis used to attach the aralkyl group to a parent molecule. Examplesinclude, but are not limited to, benzyl, phenethyl and the like. Aralkylgroups as used herein may optionally include further substitutent groupsattached to the alkyl, the aryl or both groups that form the radicalgroup.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include, but are not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstitutent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatom. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined having an alky radical that can attach theheteroarylalkyl group to a parent molecule. Examples include, but arenot limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyland the like. Heteroarylalkyl groups as used herein may optionallyinclude further substitutent groups on one or both of the heteroaryl oralkyl portions.

The term “heterocyclic radical” as used herein, refers to a radicalmono-, or poly-cyclic ring system that includes at least one heteroatomand is unsaturated, partially saturated or fully saturated, therebyincluding heteroaryl groups. Heterocyclic is also meant to include fusedring systems wherein one or more of the fused rings contain at least oneheteroatom and the other rings can contain one or more heteroatoms oroptionally contain no heteroatoms. A heterocyclic group typicallyincludes at least one atom selected from sulfur, nitrogen or oxygen.Examples of heterocyclic groups include, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and thelike. Heterocyclic groups as used herein may optionally include furthersubstitutent groups.

The term “hydrocarbyl” includes groups comprising C, O and H. Includedare straight, branched and cyclic groups having any degree ofsaturation. Such hydrocarbyl groups can include one or more heteroatomsselected from N, O and S and can be further mono or poly substitutedwith one or more substituent groups.

The term “mono or poly cyclic structure” as used herein includes allring systems that are single or polycyclic having rings that are fusedor linked and is meant to be inclusive of single and mixed ring systemsindividually selected from aliphatic, alicyclic, aryl, heteroaryl,aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic,heteroarylalkyl. Such mono and poly cyclic structures can contain ringsthat are uniform or have varying degrees of saturation including fullysaturated, partially saturated or fully unsaturated. Each ring cancomprise ring atoms selected from C, N, O and S to give rise toheterocyclic rings as well as rings comprising only C ring atoms whichcan be present in a mixed motif such as for example benzimidazolewherein one ring has only carbon ring atoms and the fused ring has twonitrogen atoms. The mono or poly cyclic structures can be furthersubstituted with substituent groups such as for example phthalimidewhich has two ═O groups attached to one of the rings. In another aspect,mono or poly cyclic structures can be attached to a parent moleculedirectly through a ring atom, through a substituent group or abifunctional linking moiety.

The term “oxo” refers to the group (═O).

The terms “bicyclic nucleic acid (BNA)” and “bicyclic nucleoside” referto a nucleoside wherein the furanose portion of the nucleoside includesa bridge connecting two carbon atoms on the furanose ring, therebyforming a bicyclic ring system.

The term “bicyclic nucleoside analog” refers to BNA like nucleosideswherein the ribose sugar has been replaced or modified. As used in thepresent application, the tetrahydropyran nucleoside analog analogs referto tetrahydropyran nucleoside analogs wherein the ribose portion of thenucleoside is replaced with a tetrahydropyran ring.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and Formulation into anefficacious therapeutic agent. Only stable compounds are contemplatedherein.

Linking groups or bifunctional linking moieties such as those known inthe art are useful for attachment of chemical functional groups,conjugate groups, reporter groups and other groups to selective sites ina parent compound such as for example an oligomeric compound. In generala bifunctional linking moiety comprises a hydrocarbyl moiety having twofunctional groups. One of the functional groups is selected to bind to aparent molecule or compound of interest and the other is selected tobind essentially any selected group such as a chemical functional groupor a conjugate group. In some embodiments, the linker comprises a chainstructure or an oligomer of repeating units such as ethylene glycols oramino acid units. Examples of functional groups that are routinely usedin bifunctional linking moieties include, but are not limited to,electrophiles for reacting with nucleophilic groups and nucleophiles forreacting with electrophilic groups. In some embodiments, bifunctionallinking moieties include amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), and the like. Somenonlimiting examples of bifunctional linking moieties include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, oligomeric compounds are modified by covalentattachment of one or more 5′ or 3′-terminal groups. The term “terminalgroup” as used herein is meant to include useful groups known to the artskilled that can be placed on one or both of the 3′ and 5′-ends of anoligomeric compound for various purposes such as enabling the trackingof the oligomeric compound (a fluorescent label or other reportergroup), improving the pharmacokinetics or pharmacodynamics of theoligomeric compound (a group for enhancing uptake and delivery) orenhancing one or more other desirable properties of the oligomericcompound (group for improving nuclease stability or binding affinity).In certain embodiments, 3′ and 5′-terminal groups include withoutlimitation, one or more modified or unmodified nucleosides, conjugategroups, capping groups, phosphate moieties and protecting groups.

In certain embodiments, oligomeric compounds are modified by covalentattachment of one or more conjugate groups. In general, conjugate groupsmodify one or more properties of the attached oligomeric compoundincluding but not limited to pharmakodynamic, pharmacokinetic, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional linking moiety or linking groupto a parent compound such as an oligomeric compound. A preferred list ofconjugate groups includes without limitation, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, thioethers,polyethers, cholesterols, thiocholesterols, cholic acid moieties,folate, lipids, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarinsand dyes.

In certain embodiments, oligomeric compounds are modified by covalentattachment of one or more 5′ or 3′-terminal groups that include but arenot limited to further modified or unmodified nucleosides. Such terminalgroups can be useful for enhancing properties of oligomeric compoundssuch as for example nuclease stability, uptake and delivery.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York(1999).

Groups can be selectively incorporated into oligomeric compounds of theinvention as precursors. For example an amino group can be placed into acompound of the invention as an azido group that can be chemicallyconverted to the amino group at a desired point in the synthesis.Generally, groups are protected or present as precursors that will beinert to reactions that modify other areas of the parent molecule forconversion into their final groups at an appropriate time. Furtherrepresentative protecting or precursor groups are discussed in Agrawal,et al., Protocols for Oligonucleotide Conjugates, Eds, Humana Press; NewJersey, 1994; Vol. 26 pp. 1-72.

Examples of hydroxyl protecting groups include, but are not limited to,acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl(TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl,pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate,mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl,dimethoxytrityl (DMT), trimethoxytrityl,1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Where more preferredhydroxyl protecting groups include, but are not limited to, benzyl,2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl,mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

Examples of amino protecting groups include, but are not limited to,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include, but are not limited to,triphenylmethyl (trityl), benzyl (Bn), and the like.

In certain embodiments, oligomeric compounds are prepared by connectingnucleosides with optionally protected phosphorus containinginternucleoside linkages. Representative protecting groups forphosphorus containing internucleoside linkages such as phosphodiesterand phosphorothioate linkages include β-cyanoethyl, diphenylsilylethyl,δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl(META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See forexample U.S. Pat. Nos. 4,725,677 and Re. 34,069 (β-cyanoethyl);Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 10, pp. 1925-1963(1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 46, pp.10441-10488 (1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron, 48 No.12, pp. 2223-2311 (1992).

The term “orthogonally protected” refers to functional groups which areprotected with different classes of protecting groups, wherein eachclass of protecting group can be removed in any order and in thepresence of all other classes (see, Barany, G. and Merrifield, R. B., J.Am. Chem. Soc., 1977, 99, 7363; idem, 1980, 102, 3084.) Orthogonalprotection is widely used in for example automated oligonucleotidesynthesis. A functional group is deblocked in the presence of one ormore other protected functional groups which is not affected by thedeblocking procedure. This deblocked functional group is reacted in somemanner and at some point a further orthogonal protecting group isremoved under a different set of reaction conditions. This allows forselective chemistry to arrive at a desired compound or oligomericcompound.

In certain embodiments, compounds having reactive phosphorus groups areprovided that are useful for forming internucleoside linkages includingfor example phosphodiester and phosphorothioate internucleosidelinkages. Such reactive phosphorus groups are known in the art andcontain phosphorus atoms in P^(III) or P^(V) valence state including,but not limited to, phosphoramidite, H-phosphonate, phosphate triestersand phosphorus containing chiral auxiliaries. A preferred syntheticsolid phase synthesis utilizes phosphoramidites (P^(III) chemistry) asreactive phosphites. The intermediate phosphite compounds aresubsequently oxidized to the P^(V) state using known methods to yield,phosphodiester or phosphorothioate internucleotide linkages. Additionalreactive phosphates and phosphites are disclosed in Tetrahedron ReportNumber 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

As used herein the term “internucleoside linkage” is meant to includeall manner of internucleoside linking groups known in the art includingbut not limited to, phosphorus containing internucleoside linking groupssuch as phosphodiester and phosphorothioate, non-phosphorus containinginternucleoside linking groups such as formacetyl and methyleneimino,and neutral non-ionic internucleoside linking groups such as amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′).

Specific examples of oligomeric compounds useful in this inventioninclude oligonucleotides containing modified e.g. non-naturallyoccurring internucleoside linkages. Two main classes of internucleosidelinkages are defined by the presence or absence of a phosphorus atom.Modified internucleoside linkages having a phosphorus atom include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Modified internucleoside linkages not having a phosphorus atom include,but are not limited to, those that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. In the context ofthis invention, the term “oligonucleoside” refers to a sequence of twoor more nucleosides that are joined by internucleoside linkages that donot have phosphorus atoms.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

As used herein the phrase “neutral internucleoside linkage” is intendedto include internucleoside linkages that are non-ionic. Neutralinternucleoside linkages include but are not limited tophosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS SymposiumSeries 580; Chapters 3 and 4, (pp. 40-65)). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH₂ component parts.

The compounds described herein can be prepared by any of the applicabletechniques of organic synthesis, as, for example, illustrated in theexamples below. Many such techniques are well known in the art. However,many of the known techniques are elaborated in Compendium of OrganicSynthetic Methods (John Wiley & Sons, New York) Vol. 1, Ian T. Harrisonand Shuyen Harrison (1971); Vol. 2, Ian T. Harrison and Shuyen Harrison(1974); Vol. 3, Louis S. Hegedus and Leroy Wade (1977); Vol. 4, Leroy G.Wade Jr., (1980); Vol. 5, Leroy G. Wade Jr. (1984); and Vol. 6, MichaelB. Smith; as well as March, J., Advanced Organic Chemistry, 3rd Edition,John Wiley & Sons, New York (1985); Comprehensive Organic Synthesis.Selectivity, Strategy & Efficiency in Modern Organic Chemistry, In 9Volumes, Barry M. Trost, Editor-in-Chief, Pergamon Press, New York(1993); Advanced Organic Chemistry, Part B: Reactions and Synthesis, 4thEd.; Carey and Sundberg; Kluwer Academic/Plenum Publishers: New York(2001); Advanced Organic Chemistry, Reactions, Mechanisms, andStructure, 2nd Edition, March, McGraw Hill (1977); Protecting Groups inOrganic Synthesis, 2nd Edition, Greene, T. W., and Wutz, P. G. M., JohnWiley & Sons, New York (1991); and Comprehensive OrganicTransformations, 2nd Edition, Larock, R. C., John Wiley & Sons, New York(1999).

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, α or β, or as (D)- or (L)- such as foramino acids. Included herein are all such possible isomers, as well astheir racemic and optically pure forms. Optical isomers may be preparedfrom their respective optically active precursors by the proceduresdescribed above, or by resolving the racemic mixtures. The resolutioncan be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds, otherunsaturation, or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

As used herein the term “oligomeric compound” is meant to include apolymer having at least a region that is capable of hybridizing to anucleic acid molecule. The term “oligomeric compound” includesoligonucleotides, oligonucleotide analogs and oligonucleosides as wellas nucleotide mimetics and/or mixed polymers comprising nucleic acid andnon-nucleic acid components and chimeric oligomeric compounds comprisingmixtures of nucleosides from any of these categories. Thetetrahydropyran nucleoside analogs can be classified as a mimetic as theribose sugar portion has been replaced with a tetrahydropyran group.Oligomeric compounds are routinely prepared linearly but can be joinedor otherwise prepared to be circular and may also include branching.Oligomeric compounds can form double stranded constructs such as forexample two strands hybridized to form double stranded compositions. Thedouble stranded compositions can be linked or separate and can includeoverhangs on the ends. In general, an oligomeric compound comprises abackbone of linked monomeric subunits where each linked monomericsubunit is directly or indirectly attached to a heterocyclic basemoiety. Oligomeric compounds may also include monomeric subunits thatare not linked to a heterocyclic base moiety thereby providing abasicsites. The linkages joining the monomeric subunits, the sugar moietiesor surrogates and the heterocyclic base moieties can be independentlymodified. The linkage-sugar unit, which may or may not include aheterocyclic base, may be substituted with a mimetic such as themonomers in peptide nucleic acids. The ability to modify or substituteportions or entire monomers at each position of an oligomeric compoundgives rise to a large number of possible motifs.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond. However, open linear structures aregenerally desired. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions. Suchnon-naturally occurring oligonucleotides are often desired overnaturally occurring forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers to asequence of nucleosides that are joined by internucleoside linkages thatdo not have phosphorus atoms. Internucleoside linkages of this typeinclude short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixedheteroatom cycloalkyl, one or more short chain heteroatomic and one ormore short chain heterocyclic. These internucleoside linkages include,but are not limited to, siloxane, sulfide, sulfoxide, sulfone, acetyl,formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl,alkeneyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate,sulfonamide, amide and others having mixed N, O, S and CH₂ componentparts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference. The term “nucleobase” or “heterocyclicbase moiety” as used herein, is intended to by synonymous with “nucleicacid base or mimetic thereof.” In general, a nucleobase or heterocyclicbase moiety is any substructure that contains one or more atoms orgroups of atoms capable of hydrogen bonding to a base of a nucleic acid.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Modified nucleobases include, but are not limited to, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theoligomeric compounds of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278).

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121;5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

In certain embodiments, oligomeric compounds may also contain one ormore nucleosides having modified sugar moieties. The furanosyl sugarring can be modified in a number of ways including substitution with asubstituent group (2′, 3′, 4′ or 5′), bridging to form a BNA andsubstitution of the 4′-O with a heteroatom such as S or N(R). Somerepresentative U.S. patents that teach the preparation of such modifiedsugars include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; 5,700,920; 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005 certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety. A representative list of preferred modified sugarsincludes but is not limited to substituted sugars having a 2′-F, 2′-OCH₂or a 2′-O(CH₂)₂—OCH₃ (2′-MOE or simply MOE) substituent group; 4′-thiomodified sugars, 4′-thio-2′-substituted sugars and bicyclic modifiedsugars.

As used herein the term “nucleoside mimetic” is intended to includethose structures used to replace the sugar or the sugar and the base notthe linkage at one or more positions of an oligomeric compound such asfor example nucleoside mimetics having morpholino or bicyclo[3.1.0]hexylsugar mimetics e.g. non furanose sugar units with a phosphodiesterlinkage. The term “sugar surrogate” overlaps with the slightly broaderterm “nucleoside mimetic” but is intended to indicate replacement of thesugar unit (furanose ring) only. The term “nucleotide mimetic” isintended to include those structures used to replace the nucleoside andthe linkage at one or more positions of an oligomeric compound such asfor example peptide nucleic acids or morpholinos (morpholinos linked by—N(H)—C(═O)—O— or other non-phosphodiester linkage).

As used herein the term “modified nucleoside” is meant to include allmanner of modified nucleosides that can be incorporated into anoligomeric compound using oligomer synthesis. The term includesnucleosides having a ribofuranose sugar and can include a heterocyclicbase but abasic modified nucleoside are also envisioned. One group ofrepresentative modified nucleosides includes without limitation bicyclicnucleosides, 2′-modified nucleosides, 4′-thio modified nucleosides and4′-thio-2′-modified nucleosides and base modified nucleosides.

As used herein the term “monomer subunit” is meant to include all mannerof monomers that can be incorporated into an oligomeric compound usingoligomer synthesis. The term includes nucleosides having a ribofuranosesugar and a heterocyclic base but also includes monomers having modifiedsugars or surrogate sugars e.g. mimetics. As such the term includesnucleosides, modified nucleosides (such as bicyclic nucleosides),nucleoside mimetics (such as the tetrahydropyran nucleoside analogsprovided herein).

Those skilled in the art, having possession of the present disclosurewill be able to prepare oligomeric compounds of essentially any viablelength to practice the methods disclosed herein. Such oligomericcompounds will include at least one and preferably a plurality oftetrahydropyranyl nucleoside analogs provided herein and may alsoinclude other monomer subunits including but not limited to nucleosides,modified nucleosides and nucleoside mimetics.

In certain embodiments, oligomeric compounds comprise from about 8 toabout 80 monomer subunits in length. One of ordinary skill in the artwill appreciate that the invention embodies oligomeric compounds of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or80 monomer subunits in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 8to 40 monomer subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 monomer subunits in length,or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 8to 20 monomer subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 monomer subunits in length, or anyrange therewithin.

In another embodiment, the oligomeric compounds of the invention are 10to 16 monomer subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 10, 11, 12,13, 14, 15 or 16 monomer subunits in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 12to 16 monomer subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 12, 13, 14,15 or 16 monomer subunits in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the invention are 10to 14 monomer subunits in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 10, 11, 12,13 or 14 monomer subunits in length, or any range therewithin.

In certain embodiments, oligomeric compounds of any of a variety ofranges of lengths of linked monomer subunits are provided. In certainembodiments, oligomeric compounds are provided consisting of X-Y linkedmonomer subunits, where X and Y are each independently selected from 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, and 50; provided that X<Y. For example, in certainembodiments, the invention provides oligomeric compounds comprising:8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20,8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11,9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23,9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14,10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24,10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15,11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25,11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17,12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27,12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20,13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30,14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24,14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19,15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29,15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25,16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22,17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20,18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30,19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-29,19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29,20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30,22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24, 23-25,23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29,24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30,27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked monomer subunits.

In certain embodiments, ranges for the length of the oligomericcompounds are 8-16, 8-40, 10-12, 10-14, 10-16, 10-18, 10-20, 10-21,12-14, 12-16, 12-18, 12-20 and 12-24 linked monomer subunits.

In certain embodiments, the ranges for the oligomeric compounds listedherein are meant to limit the number of monomer subunits in theoligomeric compounds, however such oligomeric compounds may furtherinclude protecting groups such as hydroxyl protecting groups, optionallylinked conjugate groups, 5′ and/or 3′-terminal groups and/or othersubstituents.

Chimeric oligomeric compounds have differentially modified nucleosidesat two or more positions and are generally defined as having a motifChimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Representative U.S. patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

In certain embodiments, oligomerization of modified and unmodifiednucleosides and mimetics thereof, is performed according to literatureprocedures for DNA (Protocols for Oligonucleotides and Analogs, Ed.Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23,206-217; Gait et al., Applications of Chemically synthesized RNA inRNA:Protein Interactions, Ed. Smith (1998), 1-36; Gallo et al.,Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. Additionalmethods for solid-phase synthesis may be found in Caruthers U.S. Pat.Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069.

Commercially available equipment routinely used for the support mediumbased synthesis of oligomeric compounds and related compounds is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. Suitable solid phasetechniques, including automated synthesis techniques, are described inF. Eckstein (ed.), Oligonucleotides and Analogues, a Practical Approach,Oxford University Press, New York (1991).

The synthesis of RNA and related analogs relative to the synthesis ofDNA and related analogs has been increasing as efforts in RNAi increase.The primary RNA synthesis strategies that are presently being usedcommercially include 5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-β-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries.

The primary groups being used for commercial RNA synthesis are:

-   -   TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;    -   TOM=2′-O-[(triisopropylsilyl)oxy]methyl;    -   DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl        ether-2′-O-bis(2-acetoxyethoxy)methyl    -   FPMP=5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl].

In certain embodiments, each of the aforementioned RNA synthesisstrategies can be used herein. Strategies that would be a hybrid of theabove e.g. using a 5′-protecting group from one strategy with a2′-O-protecting from another strategy are also amenable herein.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In certain embodiments,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, the target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleobases which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleobases such that stable and specificbinding occurs between the oligonucleotide and a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). In certain embodiments, oligomeric compounds cancomprise at least about 70%, at least about 80%, at least about 90%, atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an oligomeric compound in which 18 of 20nucleobases of the oligomeric compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within this scope. Percent complementarity of anoligomeric compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

Further included herein are oligomeric compounds such as antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these oligomeric compoundsmay be introduced in the form of single-stranded, double-stranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges or loops. Once introducedto a system, the oligomeric compounds of the invention may elicit theaction of one or more enzymes or structural proteins to effectmodification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded oligomeric compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of oligomeric compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

In some embodiments, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed herein in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent.

Suitable target segments may also be combined with their respectivecomplementary antisense oligomeric compounds provided herein to formstabilized double-stranded (duplexed) oligonucleotides. Such doublestranded oligonucleotide moieties have been shown in the art to modulatetarget expression and regulate translation as well as RNA processing viaan antisense mechanism. Moreover, the double-stranded moieties may besubject to chemical modifications (Fire et al., Nature, 1998, 391,806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene,2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431;Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507;Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). Forexample, such double-stranded moieties have been shown to inhibit thetarget by the classical hybridization of antisense strand of the duplexto the target, thereby triggering enzymatic degradation of the target(Tijsterman et al., Science, 2002, 295, 694-697).

The oligomeric compounds provided herein can also be applied in theareas of drug discovery and target validation. In certain embodiments,provided here is the use of the oligomeric compounds and targetsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between proteins and a disease state, phenotype, orcondition. These methods include detecting or modulating a targetpeptide comprising contacting a sample, tissue, cell, or organism withone or more oligomeric compounds provided herein, measuring the nucleicacid or protein level of the target and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther oligomeric compound of the invention. These methods can also beperformed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype. In certain embodiments, there is provided oligomericcompounds of the invention for use in therapy. In certain embodiments,the therapy is reducing target messenger RNA.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

In certain embodiments, chemically-modified oligomeric compounds of theinvention may have a higher affinity for target RNAs than doesnon-modified DNA. In certain such embodiments, higher affinity in turnprovides increased potency allowing for the administration of lowerdoses of such compounds, reduced potential for toxicity, improvement intherapeutic index and decreased overall cost of therapy.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

In certain embodiments, oligomeric compounds provided herein can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. Furthermore, antisense oligonucleotides, which areable to inhibit gene expression with exquisite specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway. In certain embodiments, oligomeric compoundsprovided herein can be utilized either alone or in combination withother oligomeric compounds or therapeutics, can be used as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of genes expressed withincells and tissues. Oligomeric compounds can also be effectively used asprimers and probes under conditions favoring gene amplification ordetection, respectively. These primers and probes are useful in methodsrequiring the specific detection of nucleic acid molecules encodingproteins and in the amplification of the nucleic acid molecules fordetection or for use in further studies. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofselected proteins in a sample may also be prepared.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more oligomeric compounds are compared to controlcells or tissues not treated with oligomeric compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compounds andor oligomeric compounds which affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

While in certain embodiments, oligomeric compounds provided herein canbe utilized as described, the following examples serve only toillustrate and are not intended to be limiting.

EXAMPLES General

¹H and ¹³C NMR spectra were recorded on a 300 MHz and 75 MHz Brukerspectrometer, respectively.

Example 1 Synthesis of Nucleoside Phosphoramidites

The preparation of nucleoside phosphoramidites is performed followingprocedures that are illustrated herein and in the art such as but notlimited to U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.

Example 2 Oligonucleoside Synthesis

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as alkylatedderivatives and those having phosphorothioate linkages.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides can be synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation is effected incertain embodiments by utilizing a 10% w/v solution of3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for theoxidation of the phosphite linkages. The thiation reaction step time isincreased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligonucleotides are recovered byprecipitating with greater than 3 volumes of ethanol from a 1 M NH₄OAcsolution. Phosphinate oligonucleotides can be prepared as described inU.S. Pat. No. 5,508,270.

Alkyl phosphonate oligonucleotides can be prepared as described in U.S.Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050.

Phosphoramidite oligonucleotides can be prepared as described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate oligonucleotides can be prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,476,925.

Phosphotriester oligonucleotides can be prepared as described in U.S.Pat. No. 5,023,243.

Borano phosphate oligonucleotides can be prepared as described in U.S.Pat. Nos. 5,130,302 and 5,177,198.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages can be prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal linked oligonucleosides can be prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide linked oligonucleosides can be prepared as described inU.S. Pat. No. 5,223,618.

Example 3 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligonucleotides or oligonucleosides arerecovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol.Synthesized oligonucleotides are analyzed by electrospray massspectroscopy (molecular weight determination) and by capillary gelelectrophoresis. The relative amounts of phosphorothioate andphosphodiester linkages obtained in the synthesis is determined by theratio of correct molecular weight relative to the −16 amu product(+/−32+/−48). For some studies oligonucleotides are purified by HPLC, asdescribed by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171.Results obtained with HPLC-purified material are generally similar tothose obtained with non-HPLC purified material.

Example 4 Oligonucleotide Synthesis 96 Well Plate Format

Oligonucleotides can be synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages are afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages are generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites are purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides are cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 5 Oligonucleotide Analysis Using 96-Well Plate Format

The concentration of oligonucleotide in each well is assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products is evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates are diluted fromthe master plate using single and multi-channel robotic pipettors.Plates are judged to be acceptable if at least 85% of the oligomericcompounds on the plate are at least 85% full length.

Example 6 Cell Culture and Oligonucleotide Treatment

The effect of oligomeric compounds on target nucleic acid expression istested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cell linesderived from multiple tissues and species can be obtained from AmericanType Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtainedfrom Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany).b.END cells were routinely cultured in DMEM, high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells wereroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 3000 cells/well for uses including but not limited tooligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated witholigomeric compounds using a transfection method as described.

LIPOFECTIN™

When cells reached 65-75% confluency, they are treated witholigonucleotide. Oligonucleotide is mixed with LIPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture isincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture is replaced with fresh culturemedium. Cells are harvested 16-24 hours after oligonucleotide treatment.

Other suitable transfection reagents known in the art include, but arenot limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, andFUGENE™. Other suitable transfection methods known in the art include,but are not limited to, electroporation.

Example 7 Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of a target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

RT and PCR reagents were obtained from Invitrogen Life Technologies(Carlsbad, Calif.). RT, real-time PCR was carried out by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RIBOGREEN™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Example 8

a) Preparation of Compound 2

Compound 1 (13.1 g, 55.9 mmol,1,5:2,3-dianhydro-4,6-O-benzylidene-D-allitol, purchased fromCarbosynth, UK), was dissolved in anhydrous N,N-dimethylformamide (210mL). To this solution was added uracil (7.52 g, 67.1 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (10.0 mL, 67.1 mmol). This mixturewas heated to 85° C. for 7 hours. The mixture was then cooled to roomtemperature, poured into ethyl acetate (1 L), and washed withhalf-saturated aqueous NaHCO₃ (4×1 L). The aqueous portion was driedover anhydrous Na₂SO₄, filtered, and evaporated to a pale foam, whichwas purified by silica gel chromatography (2% methanol in CH₂Cl₂) toyield 12.5 g (64.5% yield) of Compound 2 as a white foam. ESI-MS [M+H⁺]:calc. 347 Da; obs. 347 Da. ¹H NMR was consistent with structure.Reference for this procedure—Abramov, M.; Marchand, A.;Calleja-Marchand, A.; Herdewijn, P. Synthesis of D-Altritol Nucleosideswith a 3′-O-tert-butyldimethylsilyl protecting group. Nucleosides,Nucleotides & Nucleic Acids (2004) 23, 439.

b) Preparation of Compound 3

Compound 2 (12.1 g, 35.0 mmol) was dissolved in a mixture of anhydrousCH₂Cl₂ (50 mL) and anhydrous pyridine (50 mL). This mixture was cooledto 0° C., then treated with methane-sulfonyl chloride (6.77 mL, 87.4mmol). After maintaining at 0° C. for 15 minutes, the mixture was warmedto room temperature and stirred an additional 5 hours. Concentration invacuo yielded a golden slush, which was resuspended in CH₂Cl₂ (500 mL),washed with half-saturated aq. NaHCO₃, dried over anhydrous Na₂SO₄,filtered, and evaporated to a golden oil. Subsequent purification bysilica gel chromatography (2% methanol in CH₂Cl₂) yielded 11.7 g (78.6%yield) of Compound 3 as a pale yellow foam. ESI-MS [M+H⁺]: calc. 425 Da;obs. 425 Da. ¹H NMR was consistent with structure.

c) Preparation of Compound 4

Compound 3 (11.2 g, 26.5 mmol) was suspended in 1,4-dioxane (100 mL). Tothis suspension was added 100 mL of 2M aqueous NaOH. The resultingmixture was warmed to 60° C. and stirred for 3.5 hours. The mixture wascooled to room temperature, then neutralized with acetic acid (11.4 mL).The mixture was concentrated in vacuo to ˜100 mL and then poured intoCH₂Cl₂ (500 mL). The resulting mixture was washed with saturated aq.NaHCO₃ (500 mL), dried over anhydrous Na₂SO₄, filtered, and evaporatedto yield 8.23 g (89.7% yield) of Compound 4 as an off-white solid.ESI-MS [M+H⁺]: calc. 347 Da; obs. 347 Da. ¹H NMR was consistent withstructure.

d) Preparation of Compound 5

Compound 4 (7.96 g, 23.0 mmol) was dissolved in anhydrous THF (100 mL).To this solution was added 1,8-diazabicyclo[5.4.0]undec-7-ene (5.1 mL,34 mmol), followed by nonafluorobutanesulfonyl fluoride (11.6 mL, 34mmol), which was added dropwise with stirring. This mixture wasincubated at 30° C. for 84 hours. The mixture was poured into ethylacetate (400 mL), washed with half-saturated aq. NaHCO₃ (2×500 mL),dried over anhydrous Na₂SO₄, filtered, and evaporated to a pale foam.Silica gel chromatography (1:1 hexanes:ethyl acetate) yielded 7.92 g ofCompound 5 as an impure mixture. This mixture was used for subsequentreactions without further purification. A small portion was morecarefully purified by silica gel chromatography for analyticalcharacterization. ESI-MS [M+H⁺]: calc. 349 Da; obs. 349 Da (majorimpurity [M+H⁺]=329, consistent with elimination of HF). Both ¹H and ¹⁹FNMR were consistent with structure for Compound 5.

e) Preparation of Compound 6

Impure Compound 5 (6.87 g, 19.7 mmol) was dissolved in anhydrous CH₂Cl₂(100 mL). To this solution was added trifluoroacetic acid (35 mL). Afterstirring at room temperature for 1 hour, this mixture was concentratedin vacuo to a pale-orange oil. Purification by silica gel chromatography(stepwise gradient from 1% methanol to 10% methanol in CH₂Cl₂) yielded3.58 g (69% yield) of Compound 6 as a white foam. ESI-MS [M+H⁺]: calc.261 Da; obs. 261 Da.

f) Preparation of Compound 7

Compound 6 (3.37 g, 12.9 mmol) was dissolved in anhydrous pyridine (40mL). After cooling to 0° C., the solution was treated with4,4′-dimethoxytrityl chloride (6.59 g, 19.5 mmol). After stirring at 0°C. for 20 minutes, the mixture was warmed to room temperature for anadditional 3 hours. The resulting mixture was concentrated in vacuo to abrown oil, resuspended in CH₂Cl₂ (400 mL), washed with half-saturatedaq. NaHCO₃ (2×400 mL), dried over anhydrous Na₂SO₄, filtered, andevaporated. Silica gel chromatography (2% v/v methanol in CH₂Cl₂,yielded 5.68 g (77.9% yield) of Compound 7 as a beige foam. Both ¹H and¹⁹F NMR were consistent with structure.

g) Preparation of Compound 8

Compound 7 (2.50 g, 4.45 mmol) was dissolved in anhydrousN,N-dimethylformamide (11.2 mL). To this solution was added2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.98 mL, 6.23mmol), tetrazole (156 mg, 2.22 mmol), and N-methylimidazole (89 μL, 1.11mmol). After stirring at room temperature for 3 hours, the mixture wastreated with triethylamine (2.48 mL, 17.8 mmol), stirred for 5 minutes,then poured into ethyl acetate (250 mL). The resulting solution waswashed with 1:1 saturated aq. NaHCO₃:saturated aq. NaCl (1×200 mL),followed by saturated aq. NaCl (1×200 mL). The organic portion was driedover anhydrous Na₂SO₄, filtered, and evaporated. Silica gelchromatography (1:1 hexanes:ethyl acetate) yielded 2.61 g (76.8% yield)of Compound 8 as a pale yellow foam. ¹H, ¹⁹F, and ³¹P NMR wereconsistent with the structure of Compound 8 as a mixture of phosphorousdiastereomers.

Example 9

a) Preparation of Compound 9

Compound 7 (2.50 g, 4.44 mmol, prepared in the previous example) wasdissolved in anhydrous N,N-dimethylformamide (10 mL). To this solutionwas added imidazole (1.82 g, 26.7 mmol) and tert-butyldimethylsilylchloride (1.34 g, 8.88 mmol). After stirring at room temperature for 12hours, the mixture was poured into ethyl acetate (250 mL), washed withhalf-saturated aq. NaHCO₃ (2×200 mL) and saturated aq. NaCl (2×200 mL),dried over anhydrous Na₂SO₄, filtered, and evaporated. Silica gelchromatography (1:1 hexanes:ethyl acetate) yielded 2.52 g (83.8% yield)of Compound 9 as a white foam. ¹H and ¹⁹F NMR were consistent with theindicated structure.

b) Preparation of Compound 10

To a chilled (0° C.) suspension of 1,2,4-triazole (3.40 g, 49.2 mmol) inanhydrous acetonitrile (44 mL) was added phosphorous oxychloride (1.31mL, 14.1 mmol). After stirring at 0° C. for 20 minutes, triethylamine(9.8 mL, 70 mmol) was added to the mixture. To the resulting slurry wasadded a solution of Compound 9 (2.38 g, 3.52 mmol) in anhydrousacetonitrile (20 mL). The mixture was held at 0° C. for 1 hour, thenwarmed to room temperature for 2 hours. The mixture was subsequentlyconcentrated to approximately half its original volume, poured intoethyl acetate (250 mL), washed with half-saturated aq. NaCl (2×200 mL),dried over anhydrous Na₂SO₄, filtered, and evaporated to a yellow foam.This residue was redissolved in 1,4-dioxane (20 mL) and treated withconc. aq. NH₄OH (20 mL). The reaction vessel was sealed and stirred atroom temperature for 12 hours, at which time the mixture wasconcentrated under reduced pressure, poured into CH₂Cl₂ (200 mL), washedwith half-saturated aq. NaHCO₃ (1×200 mL), dried over anhydrous Na₂SO₄,filtered, and evaporated. Silica gel chromatography (1.5% v/v methanolin CH₂Cl₂) yielded 1.98 g (83.4%) of Compound 10 as a yellow foam.ESI-MS [M−H⁺]: calc. 674.8 Da; obs. 674.3 Da. ¹H and ¹⁹F NMR wereconsistent with structure.

c) Preparation of Compound 11

Compound 10 (1.86 g, 2.76 mmol) was dissolved in anhydrousN,N-dimethylformamide (10 mL). To the resulting solution was addedbenzoic anhydride (938 mg, 4.14 mmol). After stirring at roomtemperature for 14 hours, the mixture was poured into ethyl acetate (250mL), washed with saturated aq. NaHCO₃ (1×200 mL) and half-saturated aq.NaCl (2×200 mL), dried over anhydrous Na₂SO₄, filtered and evaporated.Silica gel chromatography (1:1 hexanes:ethyl acetate) yielded 2.12 g(98.4%) of Compound 11 as a white foam. ESI-MS [M−H⁺]: calc. 778 Da;obs. 778 Da. ¹H and ¹⁹F NMR were consistent with structure.

d) Preparation of Compound 12

Compound 11 (1.98 g, 2.54 mmol) was dissolved in anhydrous THF (3 mL).To this solution was added 3.3 mL of 1 M tetrabutylammonium fluoride inTHF. After 13 hours, the mixture was evaporated, redissolved in CH₂Cl₂,and subjected to silica gel chromatography. Elution with 1.5% (v/v)methanol in CH₂Cl₂ yielded 1.58 g (93.9%) of Compound 12 as an off-whitefoam. ESI-MS [M−H⁺]: calc. 664.7 Da; obs. 664.2 Da. ¹H and ¹⁹F NMR wereconsistent with structure.

e) Preparation of Compound 13

Compound 12 (1.52 g, 2.28 mmol) was dissolved in anhydrousN,N-dimethylformamide (5.8 mL). To this solution was added2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.00 mL, 3.19mmol), tetrazole (80 mg, 1.14 mmol), and N-methylimidazole (45 μL, 0.57mmol). After stirring at room temperature for 3 hours, the mixture wastreated with triethylamine (1.27 mL, 9.13 mmol), stirred for 5 minutes,and then poured into ethyl acetate (200 mL). The resulting solution waswashed with 1:1 saturated aq. NaHCO₃:saturated aq. NaCl (1×200 mL),followed by saturated aq. NaCl (2×200 mL). The organic portion was driedover anhydrous Na₂SO₄, filtered, and evaporated. Silica gelchromatography (1:1 hexanes:ethyl acetate) yielded 1.58 g (80.1% yield)of Compound 13 as a pale yellow foam. ¹H, ¹⁹F, and ³¹P NMR wereconsistent with the structure of Compound 13 as a mixture of phosphorousdiastereomers.

Example 10

a) Preparation of Compound 14

Compound 1 (30.0 g, 128 mmol), was dissolved in anhydrous acetonitrile(600 mL). To this solution was added thymine (48.4 g, 384 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (57.4 mL, 384 mmol). This mixture washeated to 85° C. for 12 hours. After cooling to room temperature,unreacted thymine was removed by filtration. The filtered solution wasconcentrated in vacuo to a yellow oil, redissolved in CH₂Cl₂ (500 mL),washed with saturated aqueous NaHCO₃ (2×500 mL), dried over Na₂SO₄,filtered, and concentrated to a yellow oil. Silica gel chromatography(2% methanol in CH₂Cl₂) of the dried residue yielded 30.3 g (65.6%) ofCompound 14 as an off-white foam. ¹H NMR was consistent with structure.ESI-MS [M+H⁺]: calc. 361.4 Da; obs. 361.1 Da.

b) Preparation of Compound 15

Compound 14 (30.1 g, 83.6 mmol) was dissolved in a mixture of anhydrousCH₂Cl₂ (100 mL) and anhydrous pyridine (100 mL). This mixture was cooledto 0° C., then treated with methane-sulfonyl chloride (8.4 mL, 109mmol). The mixture was kept at 0° C. for 30 minutes, then warmed to roomtemperature and stirred for an additional 24 hours. The mixture wasconcentrated in vacuo to an orange oil, which was redissolved in CH₂Cl₂(500 mL), washed with half-saturated aq. NaHCO₃ (2×500 mL), dried overanhydrous Na₂SO₄, filtered, and evaporated to a pale orange foam. ¹H NMRwas consistent with structure. ESI-MS [M+H⁺]: calc. 439.4 Da; obs. 439.1Da. The resulting material was used for subsequent reaction without anyadditional purification.

c) Preparation of Compound 16

Compound 15 (approximately 34 g crude, 78 mmol) was suspended in1,4-dioxane (125 mL). To this suspension was added 125 mL of 2M aqueousNaOH. The resulting mixture was warmed to 60° C. and stirred for 3hours. The mixture was cooled to room temperature, then neutralized withacetic acid (14 mL). The mixture was concentrated in vacuo to ˜75 mL,then poured into CH₂Cl₂ (1.75 L). The mixture was washed with saturatedaq. NaHCO₃ (2×1.5 L), dried over anhydrous Na₂SO₄, filtered andevaporated to yield a yellow solid, which was used for subsequentreaction without any additional purification. ESI-MS [M+H⁺]: calc. 361.4Da; obs. 361.1 Da. ¹H NMR was consistent with structure.

d) Preparation of Compound 17

Compound 16 (26.6 g crude, 73.8 mmol) was dissolved in anhydrous THF(450 mL). To this solution was added 1,8-diazabicyclo[5.4.0]undec-7-ene(16.5 mL, 111 mmol), followed by nonafluorobutanesulfonyl fluoride (34mL, 111 mmol), which was added dropwise with stirring. This mixture wasincubated at 30° C. for 42 hours. The resulting mixture was concentratedto ˜75 mL, then poured into EtOAc (500 mL), washed with half-saturatedaq. NaHCO₃ (2×500 mL), dried over anhydrous Na₂SO₄, filtered andevaporated to a brown oil. Silica gel chromatography (3:2 hexanes:ethylacetate) yielded 18.1 g (67.8%) of Compound 17 as an impure mixture(−82% pure by both LCMS and ¹H NMR). This mixture was used forsubsequent reactions without further purification. ESI-MS [M+H⁺]: calc.363 Da; obs. 363 Da (major impurity [M+H⁺]=343, consistent withelimination of HF).

e) Preparation of Compound 18

Impure Compound 17 (4.57 g, 12.6 mmol) was dissolved in methanol (300mL). To this solution was added Pd(OH)₂/C (9 g). Flask was flushed withH₂ gas, sealed, and maintained with an H₂ atmosphere while stirring atroom temperature. After 12 hours the H₂ gas was vented, Pd(OH)₂/C wasremoved by filtration through a celite plug, which was washed thoroughlywith additional methanol. Concentrated in vacuo to a white foam. Silicagel chromatography (5% methanol in CH₂Cl₂), yielded 10.7 g (95%) of 18as a white foam. ESI-MS [M+H⁺]: calc. 275.2 Da; obs. 275.1 Da. Both ¹HNMR and ¹⁹F NMR were consistent with structure.

f) Preparation of Compound 19

Compound 18 (10.6 g, 38.6 mmol) was dissolved in anhydrous pyridine (120mL), cooled to 0° C. and treated with 4,4′-dimethoxytrityl chloride(26.1 g, 77.2 mmol). The resulting solution was slowly warmed to roomtemperature and stirred for 14 hours. The reaction mixture was quenchedwith methanol (10 mL) and concentrated in vacuo to a brown slush. Themixture was redissolved in CH₂Cl₂ (500 mL), washed with half-saturatedaqueous NaHCO₃ (2×500 mL), dried over anhydrous Na₂SO₄, filtered andevaporated to a sticky brown foam. Silica gel chromatography (1%methanol in CH₂Cl₂) yielded 20.3 g (91%) of Compound 19 as a yellowfoam. ¹H NMR was consistent with structure.

g) Preparation of Compound 20

Compound 19 (9.00 g, 15.6 mmol) was dissolved in anhydrousN,N-dimethylformamide (37 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (7.43 mL, 23.4mmol), tetrazole (656 mg, 9.37 mmol), and N-methylimidazole (311 μL, 3.9mmol) were added. After stirring at room temperature for 3 hours, themixture was treated with triethylamine (8.7 mL, 62.4 mmol), stirred for5 minutes, then poured into ethyl acetate (500 mL). The resultingsolution was washed with half-saturated aqueous NaHCO₃ (3×500 mL), driedover anhydrous Na₂SO₄, filtered and evaporated to a sticky yellow foam.Silica gel chromatography (2:3 hexanes:ethyl acetate), followed byprecipitation from hexanes/ethyl acetate yielded 10.5 g (87% yield) ofCompound 20 as a pale yellow foam. ¹H, ¹⁹F, and ³¹P NMR were consistentwith the structure as a mixture of diastereomers.

Example 11

a) Preparation of Compound 21

Compound 19 (11.2 g, 19.4 mmol, prepared in the previous example) wasdissolved in anhydrous N,N-dimethylformamide (44 mL). To this solutionwas added imidazole (7.9 g, 116 mmol) and tert-butyldimethylsilylchloride (5.85 g, 38.8 mmol). After stirring at room temperature for 14hours, quenched with the addition of methanol (10 mL), poured into ethylacetate (500 mL), washed with half-saturated aq. NaHCO₃ (3×500 mL),dried over anhydrous Na₂SO₄, filtered, and evaporated to 13.2 g (98%) ofCompound 21 as a pale yellow foam. ¹H NMR was consistent with theindicated structure. Material was used for subsequent reaction withoutadditional purification.

b) Preparation of Compound 22

To a chilled (0° C.) suspension of 1,2,4-triazole (18.4 g, 267 mmol) inanhydrous acetonitrile (350 mL) was added phosphorous oxychloride (7.1mL, 76 mmol). After stirring at 0° C. for 30 minutes, triethylamine (53mL, 382 mmol) was added to the mixture. To the resulting slurry wasadded a solution of Compound 21 (13.2 g, 19.1 mmol) in anhydrousacetonitrile (100 mL). The mixture was held at 0° C. for 1 hour, thenwarmed to room temperature for 3.5 hours. The mixture was subsequentlyconcentrated to approximately half its original volume, poured intoethyl acetate (500 mL), washed with half-saturated aq. NaCl (2×500 mL),dried over anhydrous Na₂SO₄, filtered, and evaporated to a yellow foam.This residue was redissolved in 1,4-dioxane (175 mL) and treated withconc. aq. NH₄OH (175 mL). The reaction vessel was sealed and stirred atroom temperature for 14 hours, at which time the mixture wasconcentrated under reduced pressure, poured into CH₂Cl₂ (500 mL), washedwith half-saturated aq. NaHCO₃ (2×500 mL), dried over anhydrous Na₂SO₄,filtered, and evaporated to 12.4 g (94%) of Compound 22 as a yellowfoam, which crystallized upon drying overnight. ¹H NMR was consistentwith structure. Material was used for subsequent reaction withoutadditional purification.

c) Preparation of Compound 23

Compound 22 (12.3 g, 17.8 mmol) was dissolved in anhydrousN,N-dimethylformamide (60 mL). To the resulting solution was addedbenzoic anhydride (6.05 g, 26.7 mmol). After stirring at roomtemperature for 12 hours, the mixture was poured into ethyl acetate (500mL), washed with half-saturated aq. NaHCO₃ (3×500 mL), dried overanhydrous Na₂SO₄, filtered and evaporated. Silica gel chromatography(3:1 hexanes:ethyl acetate) yielded 13.4 g (95.1%) of Compound 23 as awhite foam. ¹H NMR was consistent with structure.

d) Preparation of Compound 24

Compound 23 (13.4 g, 16.9 mmol) was dissolved in anhydrous THF (14 mL).To this solution was added 22 mL of 1 M tetrabutylammonium fluoride inTHF. After 5 hours, the mixture was evaporated, then subjected to silicagel chromatography. Elution with 2:1 hexanes:ethyl acetate yielded 9.57g (83.2%) of Compound 24 as a white foam. ¹H NMR was consistent withstructure.

e) Preparation of Compound 25

Compound 24 (9.5 g, 14.0 mmol) was dissolved in anhydrousN,N-dimethylformamide (33 mL). To this solution was added2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (6.7 mL, 21.0mmol), tetrazole (589 mg, 8.41 mmol), and N-methylimidazole (279 μL,3.50 mmol). After stirring at room temperature for 3 hours, the mixturewas treated with triethylamine (7.8 mL, 56 mmol), stirred for 5 minutes,then poured into ethyl acetate (500 mL). The resulting solution waswashed with saturated aq. NaCl (3×500 mL), dried over anhydrous Na₂SO₄,filtered, and evaporated. Silica gel chromatography (3:1 hexanes:ethylacetate) yielded 11.8 g (95% yield) of Compound 25 as a white foam. ¹Hand ³¹P NMR were consistent with the structure of Compound 25 as amixture of phosphorous diastereomers.

Example 12

a) Preparation of Compound 27

Compound 1 (5.40 g, 4.56 mmol,1,5:2,3-dianhydro-4,6-O-benzylidene-D-allitol, purchased fromCarbosynth, UK) was mixed with 2-amino-6-chloropurine Compound 26 (5.89g, 34.69 mmol) and dried over P₂O₅ under reduced pressure overnight. Themixture was suspended in anhydrous hexamethyl phosphoramide (86 mL) and18-crown-6 (2.86 g, 10.82 mmol) and K₂CO₃ (3.46 g, 25.04 mmol) wasadded. The reaction mixture was stirred at 90° C. for 3 hours andallowed to equilibrate to room temperature. Crushed ice was added withsubsequent stirring for 1 hour. The precipitate formed was filtered andwashed with cold water followed by diethyl ether. The crude material waspurified by silica gel column chromatography eluting with 5% MeOH inCH₂Cl₂ to yield Compound 27 (7.01 g, 75%). ¹H NMR (300 MHz, DMSO-d₆) δ3.61 (m, 1H), 3.78 (t, J=10.1 Hz, 1H), 3.92 (m, 1H), 4.18-4.28 (m, 4H),5.63 (1, 1H), 5.83 (d, J=4.2 Hz, 1H), 5.40 (d, J=6.3 Hz, 1H), 5.85 (d,J=3.8 Hz, 1H), 6.99 (s, 2H), 7.31-7.42 (m, 5H), 8.21 (s, 1H); MS (ES)m/z 404.0 [M+H]⁻.

Example 13

Example 14

a) Preparation of Compound 43

Pivaloyl chloride (5.5 mmol, 0.67 mL) was added to a solution ofcommercially available 1,5-anhydro-4,6-O-benzylidene-D-glucitol(Carbosynth Limited, UK.) Compound 42 (5 mmol, 1.25 g), triethylamine(5.5 mmol, 0.77 mL) and dimethylaminopyridine (20 mg) in dichloromethane(25 mL). After stirring at room temperature for 24 hours, the reactionwas diluted with dichloromethane and washed with 5% HCl, saturatedsodium bicarbonate and brine then dried (Na2SO4) and concentrated.Purification by column chromatography (silica gel, eluting with 10 to30% ethyl acetate in hexanes) provided Compound 43 (1.06 g) and Compound44 (0.64 g) as white solids. Compound 43: ¹H NMR (300 MHz, chloroform-d)δ=7.56-7.44 (m, 2H), 7.36 (m, 3H), 5.49 (s, 1H), 4.98-4.81 (m, 1H),4.40-4.22 (m, 1H), 4.16-3.99 (m, 1H), 3.82 (s, 1H), 3.65 (s, 1H), 3.46(s, 1H), 3.41-3.27 (m, 1H), 3.27-3.15 (m, 1H), 3.04-2.80 (m, 1H),1.29-1.16 (m, 9H). Compound 44: ¹H NMR (300 MHz, chloroform-d)δ=7.49-7.40 (m, 2H), 7.39-7.32 (m, 3H), 5.53 (s, 1H), 5.08-4.91 (m, 1H),4.42-4.29 (m, 1H), 4.19-4.04 (m, 1H), 3.92-3.76 (m, 1H), 3.76-3.55 (m,2H), 3.50-3.30 (m, 2H), 1.24 (s, 9H).

b) Preparation of Compound 46

Trifluoromethanesulfonic anhydride (4.8 mmol, 0.8 mL) was added to acold (0° C.) solution of Compound 43 (3.2 mmol, 1.07 g) and pyridine(0.5 mL). After stirring for one hour the reaction was quenched byadding water and the organic layer was washed with water and brine thendried (Na₂SO₄) and concentrated to provide crude Compound 45 which wasused without any further purification. ¹H NMR (300 MHz, chloroform-d)δ=7.53-7.42 (m, 2H), 7.42-7.32 (m, 3H), 5.59 (s, 1H), 5.10 (s, 2H),4.48-4.33 (m, 1H), 4.32-4.15 (m, 1H), 3.90-3.69 (m, 2H), 3.57-3.42 (m,1H), 3.40-3.22 (m, 1H), 1.24 (s, 9H).

A solution of Compound 45 and cesium fluoride (10 mmol, 1.5 g) in t-BuOH(10 mL) was heated at 70° C. for 2 hours. The reaction was then cooledto room temperature, diluted with ethyl acetate and the organic layerwas washed with water and brine then dried (Na₂SO₄) and concentrated.Purification by column chromatography (silica gel, eluting with 10 to20% ethyl acetate in hexanes) provided Compound 46 (0.94 g, 90% from43). ¹H NMR (300 MHz, chloroform-d) δ=7.49 (m, 2H), 7.37 (m, 3H), 5.56(s, 1H), 5.29-5.02 (m, 1H), 5.02-4.81 (m, 1H), 4.49-4.32 (m, 1H),4.22-4.04 (m, 1H), 3.99-3.54 (m, 7H), 1.23 (s, 9H).

c) Preparation of Compound 49

Potassium carbonate (3.2 mmol, 0.44 g) was added to a solution ofcompound 46 (1.18 mmol, 0.4 g) in methanol (10 mL). After stirring atroom temperature for 3 hours, the solvent was evaporated under reducedpressure and the residue was partitioned between ethyl acetate andwater. The organic layer was dried (Na₂SO₄) and concentrated to provideCompound 47 which was used without any further purification. ¹H NMR (300MHz, chloroform-d) δ=7.58-7.30 (m, 5H), 5.54 (s, 1H), 5.23-4.94 (m, 1H),4.39 (dd, J=4.7, 10.0 Hz, 1H), 4.02-3.43 (m, 6H), 2.25-2.08 (m, 1H).

Trifluoromethanesulfonic anhydride (0.45 mmol, 0.08 mL) was added to acold (0° C.) solution of compound 47 (0.3 mmol, 0.08 g) and pyridine(0.05 mL). After stirring for one hour, the reaction was quenched byadding water and the organic layer was washed with water and brine thendried (Na₂SO₄) and concentrated to provide crude 49 which was usedwithout any further purification. ¹H NMR (300 MHz, chloroform-d)δ=7.58-7.32 (m, 5H), 5.55 (s, 1H), 5.28 (1H, d, J=55 Hz), 5.02-4.85 (m,1H), 4.42 (dd, J=4.9, 10.4 Hz, 1H), 4.09 (dd, J=5.7, 10.8 Hz, 1H),4.01-3.80 (m, 2H), 3.78-3.50 (m, 2H); MS (e/z), 387 (m+1).

Example 15

a) Preparation of Compound 48

Trifluoromethanesulfonic anhydride (12.0 mmol, 2.0 mL) was added to acold (0° C.) dichloromethane solution (40 mL) of Compound 42 (4.0 mmol,1.0 g) and pyridine (16 mmol., 1.3 mL). After stirring for one hour, thereaction was quenched by adding water and the organic layer was washedwith water and brine then dried and concentrated to provide crudeCompound 48 (2.24 g, quantitative) which was used without any furtherpurification. ¹H NMR (CDCl₃): δ 7.52-7.45 (m, 2H), 7.41-7.35 (m, 3H),5.58 (s, 1H), 5.08 (1H, t, J=9 Hz), 5.06-4.91 (m, 1H), 4.50-4.25 (m,2H), 3.83-3.69 (m, 2H), 3.65-3.43 (m, 2H). MS (e/z), 517 (m+1).

b) Preparation of compounds 49 and 50

Compound 48 (2.05 mmol, 1.1 g) and CsF (6.2 mmol., 0.94 g) were mixedwith dry t-butanol (15 mL) and the mixture was stirred at 90° C. for 25minutes. The reaction was cooled to room temperature and extracted withethyl acetate. The ethyl acetate solution was concentrated to drynessand the residue was purified by silica gel chromatography by elutingwith 5% ethyl acetate in hexanes. Compound 49 was obtained as clear oil(0.47 g, 59% yield). ¹H NMR (300 MHz, chloroform-d) δ=7.58-7.32 (m, 5H),5.55 (s, 1H), 5.28 (1H, d, J=55 Hz), 5.02-4.85 (m, 1H), 4.42 (dd, J=4.9,10.4 Hz, 1H), 4.09 (dd, J=5.7, 10.8 Hz, 1H), 4.01-3.80 (m, 2H),3.78-3.50 (m, 2H); MS (e/z), 387 (m+1). Compound 50 was obtained as awhite solid (0.14 g, 18% yield). ¹H NMR (CDCl₃): δ 7.50-7.43 (m, 2H),7.40-7.34 (m, 3H), 5.64 (s, 1H), 5.15-4.90 (m, 2H), 4.45-4.15 (m, 3H),3.80-3.52 (m, 2H), 3.55-3.40 (m, 1H). MS (e/z), 387 (m+1).

Example 16

a) Preparation of Compound 51

NaH (1.3 mmol, 52 mg) was added to a cold (0° C.) solution of Compound47 (1.0 mmol, 0.27 g) and 2-(bromomethyl)naphthalene (1.3 mmol, 0.28 g)in dimethylformamide (5 mL). After stirring for one hour, the reactionwas quenched by adding water and the mixture was extracted with ethylacetate. The ethyl acetate solution was washed with water and brine thendried and concentrated to provide crude Compound 51 which was purifiedby silica gel column chromatography by eluting with 5% ethyl acetate inhexanes. Compound 51 was obtained as a white solid (0.4 g,quantitative). ¹H NMR (CDCl₃): δ 8.0-7.25 (m, 12H), 5.47 (s, 1H), 5.17(1H, d, J=54 Hz), 4.87-4.76 (m, 2H), 4.40-4.30 (m, 1H), 3.95-3.78 (m,2H), 3.75-3.56 (m, 2H), 3.51-3.39 (m, 2H). MS (e/z), 395, 417 (m+l,m+23).

b) Preparation of Compound 52

Molecular sieves 4A (powder, 4.45 g) were placed in a 100 mL flask withheating at 140° C. over four hours with vacuation. After cooling to roomtemperature, Compound 51 and dichloromethane (15 mL) were added. Afterstirring for one hour at room temperature, the mixture was cooled to−78° C., and Et₃SiH (4.11 mmol. 0.66 mL) and PhBCl₃ (3.63 mmol. 0.48 mL)were added successively with constant stirring. The mixture was stirredfor an additional 10 minutes at −78° C. and 30% H₂O₂ (12.6 mmol. 1.6 mL)was added. After filtration, the reaction mixture was extracted withdichloromethane. The organic solution was washed with water and brinethen dried and concentrated to provide crude Compound 52 which waspurified by silica gel column chromatograph by eluting with 1% acetonein dichloromethane. Compound 52 was obtained as a white solid (0.31 g,62%). ¹H NMR (CDCl₃): δ 7.87-7.77 (m, 4H), 7.52-7.46 (m, 3H), 7.40-7.30(m, 5H), 5.14 (1H, d, J=54 Hz), 4.83-4.52 (m, 4H), 3.90-3.83 (m, 2H),3.73-3.66 (m, 3H), 3.56-3.34 (m, 2H), 1.68 (1H, t, J=6 Hz). MS (e/z),419 (m+23).

c) Preparation of Compound 53

Compound 52 (0.025 mmol. 0.01 g) was dissolved in dichloromethane (0.3mL), Dess-Martin reagent (0.025 mmol. 0.01 g) was added. The reactionwas stirred at room temperature for 10 minute and concentrated toprovide Compound 53. ¹H NMR (CDCl₃): δ 9.70 (s, 1H), 8.1-7.3 (m, 12H),5.17 (1H, d, J=54 Hz), 4.80 (s, 2H), 4.45-4.75 (m, 2H), 4.25-4.20 (m,1H), 4.0-3.90 (m, 1H), 3.85-3.35 (m, 3H).

d) Preparation of Compound 58a

Compounds 54a and 54b are prepared from Compound 53 by adding MeMgBr inthe presence of Cerium chloride. Alternately, compounds 54a and 54b canbe interconverted to each other by means of a Mitsunobu reaction. Thesecondary hydroxyl group in 54a is protected as an ester, preferably asan isobutyryl ester and the 2′O-naphthyl group is removed using DDQfollowed by reaction with triflic anhydride to provide Compound 55a.Reaction with a suitably protected nucleobase and a strong base such assodium hydride in a solvent such as DMSO at temperatures between 50 and100° C., followed by removal of the benzyl group using catalytichydrogenation and reprotection as the silyl ether provides Compound 56a.Removal of the isobutyryl group using methanolic ammonia or potassiumcarbonate in methanol followed by reaction with DMTCl and lutidine andpyridine as the solvent at temperatures between 25 and 50 degree celciusfollowed by removal of the silyl protecting group using triethylaminetrihydrofluoride provides Compound 57a. A phosphitylation reactionprovides the phosphoramidite, Compound 58a.

Example 17

Compounds 54a and 54b are prepared from aldehyde 53 by adding MeMgBr inthe presence of Cerium chloride. Alternately, compounds 54a and 54b canbe interconverted to each other by means of a Mitsunobu reaction. Thesecondary hydroxyl group in 54b is protected as an ester, preferably asan isobutyryl ester and the 2′O-naphthyl group is removed using DDQfollowed by reaction with triflic anhydride to provide Compound 55b.Reaction with a suitably protected nucleobase and a strong base such assodium hydride in a solvent such as DMSO at temperatures between 50 and100° C., followed by removal of the benzyl group using catalytichydrogenation and reprotection as the silyl ether provides Compound 56b.Removal of the isobutyryl group using methanolic ammonia or potassiumcarbonate in methanol followed by reaction with DMTCl and lutidine andpyridine as the solvent at temperatures between 25 and 50 degree Celsiusfollowed by removal of the silyl protecting group using triethylaminetrihydrofluoride provides Compound 57b. A phosphitylation reactionprovides phosphoramidite 58b.

Example 18

a) Preparation of Compound 59

Compound 53 (0.7 mmol. 0.27 g) was dissolved in THF (2 mL), water (0.7mL), HCHO (0.7 mL), and 4 N NaOH (aq., 0.7 mL) was added. The reactionwas stirred at room temperature for three days. The reaction wasextracted with ethyl acetate and washed with water and brine then driedand concentrated to provide crude 59 which was purified by silica gelcolumn chromatograph by eluting with 10% acetone in dichloromethane.Compound 59 was obtained as a white solid (0.19 g, 64%). ¹H NMR (CDCl₃):7.94-7.80 (m, 4H), 7.61-7.45 (m, 3H), 7.42-7.21 (m, 5H), 5.20 (1H, d,J-54 Hz), 4.49-4.40 (m, 4H), 4.20-3.35 (m, 11H), 2.10-1.95 (m, 1H),1.90-1.75 (m, 1H).

b) Preparation of Compound 63

Reaction of Compound 59 with TBDPSCl provides a mixture of monosilylated products which are separated and the hydroxyl group isdeoxygenated by means of a Barton deoxygenation reaction to provideCompound 60. Removal of the 2′O-naphthyl group with DDQ followed bytriflation and reaction with a suitably protected nucleobase and astrong base such as sodium hydride in a solvent such as DMSO attemperatures between 50 and 100° C. provides Compound 61. Removal of thesilyl protecting group using triethylamine trihydrofluoride followed byremoval of the benzyl group by catalytic hydrogenation provides Compound62. Protection of the primary hydroxyl group as the DMT ether followedby a phosphitylation reaction provides the phosphoramidite, Compound 63.

Example 19

Compound 65 is prepared from known Compound 64 according to the methoddescribed by Bihovsky (J. Org. Chem., 1988, 53, 4026-4031). The benzylprotecting groups are removed using catalytic hydrogenation followed byprotection of the 4′-OH and the 6′-OH as the benzylidene acetal.Reaction with triflic anhydride provides the his triflate 66. Selectivedisplacement of the 3′-triflate group using CsF as described in Example15, followed by heating with a suitably protected nucleobase in thepresence of a strong base like sodium hydride and a polar solvent likedimethyl-sulfoxide at temperatures between 50 and 100 degree Celsius andremoval of the benzylidene protecting group using aqueous acetic acid attemperatures between 50 to 100 degree Celsius provides the nucleoside67. Reaction of the primary alcohol with DMTCl followed by aphosphitylation reaction provides the phosphoramidite, Compound 68.

Example 20

Compound 69 is prepared by reacting commercially availableMethyl-β-D-glucopyranose with dimethylbenzylidene acetal in the presenceof p-toluenesulfonic acid at temperatures between 60 and 80 degreeCelsius. Selective protection of Compound 69 with pivaloyl chloride,triflation, displacement with CsF and hydrolysis of the pivaloyl esterwith potassium carbonate in methanol as described in Example 14 providesCompound 70. Removal of the benzylidene protecting group followed byreprotection of the hydroxyl groups as the benzyl ether providesCompound 71. Hydrolysis of the OMe acetal by heating with acetic acidand aqueous sulfuric acid followed by oxidation of the lactol withacetic anhydride in DMSO and an olefination reaction with Tebbe's orPetassis's reagent provides the olefin 72. Reduction of the vinyl groupand removal of the benzyl protecting groups using catalytichydrogenation followed by reprotection of the 4′OH and the 6′OH as thebenzylidene acetal provides Compound 73. Triflation with triflicanhydride followed by reaction with a suitably protected nucleobase anda strong base such as sodium hydride in a solvent such as DMSO attemperatures between 50 and 100° C. provides Compound 74. Removal of thebenzylidene protecting group using catalytic hydrogenation, protectionof the primary alcohol as the DMT ether and a phosphitylation reactionprovides the phosphoramidite Compound 75.

Example 21

Compound 76 is prepared according to the procedure described by Houlton(Tetrahedron, 1993, 49, 8087) and is reduced to Compound 77 by means ofa catalytic hydrogenation reaction. Protection of the 4′OH and the 6′OHas the benzylidene acetal provides Compound 78. Treatment of the 2′OHwith pivaloyl chloride according to method described in Example 14followed by Barton deoxygenation of the 3′OH group and hydrolysis of thepivaloyl ester provides Compound 79. Triflation with triflic anhydridefollowed by reaction with a suitably protected nucleobase and a strongbase such as sodium hydride in a solvent such as DMSO at temperaturesbetween 50 and 100° C. provides Compound 80. Removal of the benzylideneprotecting group using catalytic hydrogenation, protection of theprimary alcohol as the DMT ether and a phosphitylation reaction providesthe phosphoramidite, Compound 81.

Example 22

Oxidation of Compound 43 (prepared as per the procedures illustrated inExample 14) followed by a Wittig reaction provides Compound 82.Reduction of the olefin by means of a catalytic hydrogenation reactionfollowed by removal of the pivaloyl group with potassium carbonate inmethanol provides Compound 83. Triflation with triflic anhydridefollowed by reaction with a suitably protected nucleobase and a strongbase such as sodium hydride in a solvent such as DMSO at temperaturesbetween 50 and 100° C. provides Compound 84. Removal of the benzylideneprotecting group using catalytic hydrogenation, protection of theprimary alcohol as the DMT ether and a phosphitylation reaction providesthe phosphoramidite, Compound 85.

Example 23

Compound 45 (prepared as per the procedures illustrated in Example 14)is reacted with a suitable nucleophile such as sodium azide, sodiumcyanide, sodium sulfide, a primary or secondary amine derivative orsodium methoxide provides Compound 86 wherein the nucleophile (Nu) canbe selected from any desired nucleophile which can include suchnucleophiles as azide, cyanide, thiol, thioether, amine or alkoxide.Hydrolysis of the pivaloyl group using potassium carbonate providesCompound 87. Triflation of the hydroxyl group using triflic anhydrideprovides Compound 88. Reaction with a suitably protected nucleobase anda strong base such as sodium hydride in a solvent such as DMSO attemperatures between 50 and 100° C. provides Compound 89. Removal of thebenzylidene protecting group using catalytic hydrogenation or by heatingwith aqueous acetic acid provides Compound 90. Protection of the primaryalcohol as the DMT ether provides Compound 91 followed by aphosphitylation reaction provides the phosphoramidite, Compound 92.

Example 24

Compound 45 is treated with potassium acetate and 18-crown-6 in anappropriate solvent to afford S_(N)2 substitution of the triflate. Theresulting product is treated with methanolic ammonia at reducedtemperature to afford Compound 93. Alternately, Compound 45 can besubjected to Mitsunobu conditions (R₃P, DIAD, pO₂NBzOH), followed byaminolysis, to afford the same Compound 93. Sequential treatment of 93with triflic anhydride, isolation of the triflate, and treatment withcesium fluoride in t-butyl alcohol gives 94, analogous to thepreparation of Compound 46 from Compound 45 described above. Treatmentof 94 with potassium carbonate in methanol generates the fluoro alcohol95, which is converted to the triflate upon treatment with triflicanhydride in pyridine. Isolation, followed by treatment with anucleobase in the presence of a strong base such as sodium hydride givesCompound 96. Removal of the benzylidene protecting group with 90%aqueous acetic acid gives Compound 97. Reaction with4,4′-dimethoxytrityl chloride in pyridine gives Compound 98, which,following isolation, is converted to the cyanoethyl phosphoramidite,Compound 99.

Example 25

Oxidation of Compound 43 (prepared as per the procedures illustrated inExample 14) under Swern conditions (oxalyl chloride, DMSO,triethylamine, dichloromethane) gives ketone 100. Treatment with afluorinating reagent such as 1,1,2,2-tetrafluoroethyl-N,N-dimethylamine(alternately deoxofluor or DAST) gives Compound 101. Removal of thepivaloyl group under potassium carbonate/methanol conditions givesCompound 102. Sequential treatment with triflic anhydride in pyridine,isolation, and treatment with a nucleobase in the presence of base givesthe nucleoside analog, Compound 103. Removal of the benzylidene with 90%aqueous acetic acid gives Compound 104, which is converted to Compound105 upon treatment with 4,4-dimethoxytrityl chloride in pyridine. Aphosphitylation reaction provides the phosphoramidite, Compound 106.

Example 26

Treatment of Compound 42 (prepared as per the procedures illustrated inExample 14) with 2-(bromomethyl)-naphthalene (Nap bromide) in thepresence of sodium hydride gives a mixture of Nap-protected regioisomers(107 and 108). Separation by silica gel chromatography provides theisomer, Compound 107. Oxidation of Compound 107 under Swern conditions(oxalyl chloride, DMSO, triethylamine, dichloromethane) gives theketone, Compound 109, which is subsequently treated with methylmagnesium bromide (Methyl Grignard) to give a mixture of the methylalcohols, compounds 110 and 111. Isolation of the desired stereoisomer110 by silica gel chromatography, followed by formation of the triflateunder triflic anhydride/pyridine conditions and treatment with cesiumfluoride gives the fluorinated Compound 112. Alternatively, treatment of110 with TFEDMA gives Compound 112 in a single process. Removal of theNap protecting group with DDQ, followed by triflation, isolation, andtreatment with a nucleobase in the presence of a base gives Compound113. Removal of the benzylidene with 90% aqueous acetic acid affordsCompound 114, which is converted to Compound 115 upon treatment with4,4-dimethoxytrityl chloride in pyridine. A phosphitylation reactionprovides the phosphoramidite, Compound 116.

Example 27

Treatment of Compound 42 (prepared as per the procedures illustrated inExample 14) with tertbutyldimethylsilyl chloride in the presence ofimidazole and DMF yields a mixture of the silylated compounds 117 and118 as described previously in Nucleosides, Nucleotides, and NucleicAcids (2004), 23(1&2), 439-455. Following silica gel chromatography, theisomer, Compound 117 is oxidized under Swern conditions (oxalylchloride, DMSO, triethylamine, dichloromethane) to generate the ketone,Compound 119. Treatment with methyl magnesium bromide gives a mixture ofalcohols, compounds 120 and 121. Separation by silica gelchromatography, treatment of isolated Compound 120 withtetrabutylammonium fluoride, followed by conversion to the tosylateunder tosyl chloride and pyridine conditions, gives Compound 122.Treatment with base converts tosylate 122 to the corresponding epoxide,Compound 123, as documented with similar compounds (Bioorg. Med. Chem.Lett. 1996, 6, 1457). Reaction of Compound 123 with a selectedpyrimidine heterocycle (heterocyclic base) in the presence of baseresults in formation of Compound 124. Inversion of stereochemistry ofthe hydroxyl group is achieved by treatment with mesyl chloride,followed by hydrolysis of the resulting mesylate, which proceeds throughan anhydro cyclic intermediate. Fluorination with nonafluorobutanesulfonyl fluoride under DBU/THF conditions gives the fluorinatedCompound 126. Removal of the benzylidene group with 90% aqueous aceticacid affords Compound 127, which is converted to Compound 128 upontreatment with 4,4-dimethoxytrityl chloride in pyridine. Aphosphitylation reaction provides the phosphoramidite, Compound 129.

Example 28

a) Preparation of Compound 130

Compound 49 (prepared as per the procedures illustrated in Example 14,10.8 mmol, 4.20 g) and adenine (54.5 mmol, 7.35 g) were suspended inanhydrous DMSO (80 mL). To this suspension was added sodium hydride(54.4 mmol, 2.18 g of a 60% mineral oil suspension). The resultingmixture was heated to 55° C. for 12 hours, cooled to room temperatureand poured into water (400 mL). The mixture was extracted with ethylacetate (3×400 mL), and the combined organic extracts were washed withhalf-saturated aqueous NaCl (3×500 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered, and evaporated to give 3.93 g (97% yield) ofa brown solid. NMR (¹H and ¹⁹F) and LCMS mass analysis were consistentwith structure. This material was used without further purification.

b) Preparation of Compound 131

Compound 130 (10.5 mmol, 3.93 g) was dissolved in anhydrous pyridine (50mL). After cooling to 0° C., the solution was treated with benzoylchloride (16.9 mmol, 1.97 mL). Stirring was continued at 0° C. for 15minutes at which time the mixture was warmed to room temperature over2.5 hours. The mixture was cooled to 0° C., quenched with 20 mL H₂O andstirred for 15 minutes. Concentrated aqueous NH₄OH (20 mL) was added tothe mixture with stirring for 30 minutes. The mixture was concentratedmixture in vacuo to approximately 40 mL and poured into ethyl acetate(500 mL). The mixture was washed with half-saturated aqueous NaCl (3×500mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to alight-brown foam. Purification by silica gel chromatography (1.5%methanol in dichloromethane) yielded 2.33 g of Compound 131 as a lightbrown foam. NMR (¹H and ¹⁹F) and LCMS analyses were consistent withstructure.

c) Preparation of Compound 132

Compound 131 (4.84 mmol, 2.30 g) was dissolved in 70 mL of 90% (v/v)aqueous acetic acid. The solution was heated to 80° C. for 4 hours andthen concentrated in vacuo to a viscous yellow oil. Triethylamine (10drops) were added followed by 5 mL of methanol and 100 mL ethyl acetate.A white precipitate formed, which was collected by filtration, washedwith ethyl acetate, and vacuum dried overnight. Final mass of whitesolid, Compound 132, was 1.28 g (69%). NMR (¹H and ¹⁹F) and LCMSanalyses were consistent with structure of Compound 132.

d) Preparation of Compound 133

Compound 132 (3.24 mmol, 1.25 g) was suspended in anhydrous pyridine (12mL). The resulting suspension was cooled to 0° C. and treated with4,4′-dimethoxytrityl chloride (5.19 mmol, 1.76 g) with stirring.Stirring was continued at 0° C. for 15 minutes and at room temperaturefor 5 hours when the mixture was quenched with methanol (2 mL) andconcentrated in vacuo to a thick yellow oil. The oil was dissolved indichloromethane (150 mL) and washed with saturated aqueous NaHCO₃ (100mL) followed by saturated aqueous NaCl (2×100 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered, and evaporated to a yellow foam.Purification by silica gel chromatography yielded 2.05 g (92% yield) ofCompound 133 as a yellow foam. NMR analysis (¹H and ¹⁹F) was consistentwith structure.

e) Preparation of Compound 134

Compound 133 (2.59 mmol, 1.79 g) was dissolved in anhydrous DMF (6 mL)tetrazole (1.56 mmol, 109 mg), 1-methylimidazole (0.65 mmol, 52 μL) andtetraisopropylamino-2-cyanoethylphosphorodiamidite (3.90 mmol, 1.24 mL)were added. After stirring for 4.5 hours, the reaction was quenched withthe addition of triethylamine (10.4 mmol, 1.45 mL). The mixture waspoured into ethyl acetate (150 mL), washed with saturated aqueous NaCl(4×100 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to apale yellow foam. The solid was redissolved in ethyl acetate (7 mL) andprecipitated by dropwise addition into 70 mL of hexanes. Silica gelpurification (1:1 hexanes:ethyl acetate) of the resulting precipitateyielded 1.92 g (83%) of Compound 134 as a white foam. NMR (¹H, ¹⁹F, and³¹P) are consistent with structure. ³¹P NMR (CDCl₃): δ ppm 151.64,151.58, 150.37, 150.33.

Example 29

a) Preparation of Compound 135

Compound 49 (prepared as per the procedures illustrated in Example 14,7.51 mmol, 2.9 g) and 6-iodo-2-aminopurine tetrabutylammonium salt (17.6mmol, 8.5 g, prepared as described in J. Org. Chem. 1995, 60,2902-2905), were dissolved in anhydrous HMPA (26 mL). The mixture wasstirred at room temperature for 18 hours, poured into ethyl acetate,washed with water and saturated NaCl, dried over anhydrous Na₂SO₄,filtered and evaporated. Purification by silica gel chromatography (1:1hexanes:ethyl acetate) yielded 2.78 g (75% yield) of Compound 135. NMR(¹H and ¹⁹F) and LCMS analyses were consistent with structure.

b) Preparation of Compound 136

Compound 135 (0.64 mmol, 0.32 g) was dissolved in 1,4-dioxane (9 mL) and9 mL of 1M aqueous NaOH was added with heating at 55° C. for 18 hours.The mixture was cooled then neutralized with 1N HCl. The mixture wasconcentrated in vacuo and the residue purified by silica gelchromatography (5% methanol in dichloromethane) to yield 0.22 g (88%yield) of 136. NMR (¹H and ¹⁹F) and LCMS analyses were consistent withstructure.

c) Preparation of Compound 137

Compound 136 (3.23 mmol, 1.25 g) was dissolved in anhydrous pyridine(13.6 mL), cooled to 0° C., then treated with isobutyryl chloride (4.85mmol, 0.51 mL). The mixture was warmed to room temperature and stirredfor 6 hours. The mixture was cooled to 0° C. and treated withconcentrated aqueous NH₄OH (3.2 mL) with stirring for 30 minutes. Themixture was poured into ethyl acetate (100 mL), washed with water (200mL) and brine (200 mL), dried over anhydrous Na₂SO₄, filtered, andevaporated. Purification by silica gel chromatography (gradient of 0 to5% methanol in dichloromethane) yielded 1.21 g (82% yield) of Compound137. NMR (¹H and ¹⁹F) and LCMS analyses were consistent with structure.

d) Preparation of Compound 138

Compound 137 (0.219 mmol, 0.103 g) was dissolved in methanol (10 mL) andacetic acid (0.2 mL) and Pd(OH)₂/C (0.44 g) were added with stirringunder an atmosphere (balloon pressure) of hydrogen for 14 hours. Thecatalyst was removed by filtration, and the resulting filtrate wasconcentrated and triturated with acetonitrile to obtain Compound 138 asa white solid. NMR (¹H and ¹⁹F) and LCMS analyses were consistent withstructure.

e) Preparation of Compound 139

Compound 138 (3.83 mmol, 1.41 g) was dissolved in anhydrous pyridine (32mL) and 4,4′-dimethoxytrityl chloride (5.0 mmol, 1.71 g) was added withstirring at room temperature for 3 hours followed by quenching withmethanol (0.5 mL). The solution was concentrated in vacuo, thenredissolved in ethyl acetate. The organic solution was washed withsaturated aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄,filtered, and evaporated. Purification by silica gel chromatographyyielded 1.63 g (70% yield) of 139. NMR (¹H and ¹⁹F) analysis wasconsistent with structure.

f) Preparation of Compound 140

Compound 139 (1.59 mmol, 1.07 g) was dissolved in anhydrous DMF (4.25mL) and tetrazole (1.35 mmol, 95 mg), 1-methylimidazole (0.45 mmol, 35μL), and tetraisopropyl-2-cyanoethylphosphorodiamidite (2.25 mmol, 0.71mL) were added. The mixture was stirred at room temperature for 3 hours,poured into ethyl acetate and washed with saturated aqueous NaHCO₃ andbrine. The organic layer was dried over anhydrous Na₂SO₄, filtered, andevaporated. Purification by silica gel chromatography yielded 1.07 g(78% yield) of Compound 140. NMR (¹H, ¹⁹F, and ³¹P) analysis wasconsistent with structure. ³¹P NMR (CDCl₃): δ ppm 151.30, 151.24,148.82, 148.78.

Example 30 Preparation of Gapped Oligomeric Compounds

Automated solid-phase synthesis was used to prepare oligomeric compoundsused herein. One illustrative gapped oligomeric compound is ISIS-410131,having SEQ ID NO: 01, and Formula: 5′-C_(f)U_(f)TAGCACTGGCC_(f)U_(f)-3′.Each internucleoside linking group is a phosphorothioate, each of the T,A, G and C letters not followed by a subscript f designates aβ-D-2′-deoxyribonucleoside and each C_(f) and U_(f) is a monomer subunitwherein Bx is the heterocyclic base cytosine or uridine respectively andwherein the monomer subunit has the Formula and configuration:

The synthesis of 410131 was carried out on a 40 μmol scale using an ÄKTAOligopilot 10 (GE Healthcare) synthesizer with a polystyrene solidsupport loaded at 200 mmol/g with a universal linker. All nucleosidephosphoramidites, including compounds 8 and 13 were prepared as 0.1 Msolutions in anhydrous acetonitrile. Coupling was performed using 4molar equivalents of the respective phosphoramidite in the presence of4,5-dicyanoimidazole, with a coupling time of 14 minutes. Thiolation oftrivalent phosphorous to the phosphorothioate was achieved upontreatment with 0.2 M phenylacetyl disulfide in 1:13-picoline:acetonitrile. The resulting gapped oligomeric compound wasdeprotected using 1:1 triethylamine:acetonitrile (1 hour at roomtemperature), followed by conc. aq. NH₄OH at 55° C. for 7 hours. Ionexchange purification followed by reverse-phase desalting yielded 9.8μmol (44 mg) of purified oligonucleotide. Mass and purity analysis byLC/MS ion-pair chromatography showed a UV purity of 98.5%, with an ESImass of 4522.8 Da (calc. 4523.6 Da).

Example 31 2-10-2 Gapped Oligomeric Compounds Targeted to PTEN In VitroStudy

Gapped oligomeric compounds were synthesized and tested for theirability to reduce PTEN expression over a range of doses. bEND cells weretransfected with gapped oligomeric compounds at doses of 0.3125, 0.625,1.25, 2.5, 5, 10, 20 or 40 nM using 3 μg/mL Lipofectin in OptiMEM for 4hrs, after which transfection mixtures were replaced with normal growthmedia (DMEM, high glucose, 10% FBS, pen-strep). RNA was harvested thefollowing day (approximately 24 hours from the start of transfection)and analyzed for PTEN and cyclophilin A RNA levels using real timeRT-PCR. Values represent averages and standard deviations (n=3) of PTENRNA levels normalized to those of cyclophilin A.

The resulting dose-response curves were used to determine the IC₅₀slisted below. Tms were determined in 100 mM phosphate buffer, 0.1 mMEDTA, pH 7, at 260 nm using 4 μM of the modified oligomers listed belowand 4 μM of the complementary RNA AGGCCAGTGCTAAG (SEQ ID NO: 7).

SEQ ID NO./ Composition Tm IC₅₀ ISIS NO. (5′ to 3′) (° C.) (nM)01/392753 C_(e)U_(e)TAGCACTGGCC_(e)U_(e) 51.3 37 01/410312C_(m)U_(m)TAGCACTGGCC_(m)U_(m) 49.2 23 01/410131C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 50.0 16

Each internucleoside linking group is a phosphorothioate. Subscriptednucleosides are defined below wherein Bx is a heterocyclic base:

Example 32 2-10-2 Gapped Oligomeric Compounds Targeted to PTEN In VitroStudy

Gapped oligomeric compounds were synthesized and tested for theirability to reduce PTEN expression over a range of doses. bEND cells weretransfected with gapped oligomeric compounds at doses of 0.3125, 0.625,1.25, 2.5, 5, 10, 20 or 40 nM using 3 μg/mL Lipofectin in OptiMEM for 4hrs, after which transfection mixtures were replaced with normal growthmedia (DMEM, high glucose, 10% FBS, pen-strep). RNA was harvested thefollowing day (approximately 24 hours from start of transfection) andanalyzed for PTEN and cyclophilin A RNA levels using real time RT-PCR.Values represent averages and standard deviations (n=3) of PTEN RNAlevels normalized to those of cyclophilin A.

SEQ ID NO./ Composition ISIS NO. (5′ to 3′) 02/392063^(Me)C_(l)T_(l)TAGCACTGGC^(Me)C_(l)T_(l) 01/410131C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 02/417999^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f)

SEQ ID NO./ % UTC @ Dosage ISIS NO. 0.3125 0.625 1.25 2.5 5 10 20 4002/392063 86 83 66 40 36 24 32 17 01/410131 78 70 71 50 52 35 29 1702/417999 98 108 77 72 68 43 33 20

Each internucleoside linking group is a phosphorothioate and superscriptMe indicates that the following C is a 5-methyl C. Subscriptednucleosides are defined below wherein Bx is a heterocyclic base:

Example 33 2-10-2 Gapped Oligomeric Compounds Targeted to PTEN In VivoStudy

Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected once with the gapped oligomeric compounds targeted to PTEN at adose of 20 or 60 mg/kg. The mice were sacrificed 72 hrs followingadministration. Liver tissues were homogenized and mRNA levels werequantitated using real-time PCR as described herein for comparison tountreated control levels (% UTC). Plasma chemistry analysis wascompleted.

SEQ ID NO./ Composition dose % ISIS NO. (5′ to 3′) (mg/kg) UTC salineN/A 100 01/392753 C_(e)U_(e)TAGCACTGGCC_(e)U_(e) 20  84 01/392753C_(e)U_(e)TAGCACTGGCC_(e)U_(e) 60  68 01/410312C_(m)U_(m)TAGCACTGGCC_(m)U_(m) 20  83 01/410312C_(m)U_(m)TAGCACTGGCC_(m)U_(m) 60  27 01/410131C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 20  26 01/410131C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 60  8

Each internucleoside linking group is a phosphorothioate. Subscriptednucleosides are defined below:

No increase in ALT and no significant effect on body or organ weightswere observed after treatment with these gapped oligomeric compounds.

Example 34 Gapped Oligomeric Compounds Targeted to PTEN In Vivo Study

Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected twice per week for three weeks with the gapped oligomericcompounds targeted to PTEN at a dose of 0.47, 1.5, 4.7 or 15 mg/kg. Themice were sacrificed 48 hours following last administration. Livertissues were homogenized and mRNA levels were quantitated usingreal-time PCR as described herein for comparison to untreated controllevels (% UTC). Plasma chemistry analysis was completed. Tms weredetermined in 100 mM phosphate buffer, 0.1 mM EDTA, pH 7, at 260 nmusing 4 μM of the modified oligomers listed below and 4 μM of thecomplementary RNA AGGCCAGTGCTAAG (SEQ ID NO: 7).

SEQ ID NO./ Composition Tm ISIS NO. (5′ to 3′) (° C.) 01/410131C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 50.7 02/417999^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f) 52.6

Each internucleoside linking group is a phosphorothioate, superscript Meindicates that the following C is a 5 methyl C and nucleosides followedby a subscript f are defined in the formula below wherein Bx is aheterocyclic base:

SEQ ID NO./ % UTC @ % UTC @ % UTC @ % UTC @ ISIS NO. 0.47 mg/kg 1.5mg/kg 4.7 mg/kg 15 mg/kg 01/410131 — — — 12 02/417999 77 64 31 10 Saline% UTC = 100 (dosage N/A)

Liver transaminase levels, alanine aminotranferease (ALT) and aspartateaminotransferase (AST), in serum were also measured relative to salineinjected mice. The approximate liver transaminase levels are listed inthe table below.

SEQ ID NO./ AST @ AST @ AST @ AST @ ISIS NO. 0.47 mg/kg 1.5 mg/kg 4.7mg/kg 15 mg/kg 01/410131 — — — 106 02/417999 51 90 86 37 Saline 82(dosage N/A) SEQ ID NO./ ALT @ ALT @ ALT @ ALT @ ISIS NO. 0.47 mg/kg 1.5mg/kg 4.7 mg/kg 15 mg/kg 01/410131 — — — 27 02/417999 28 31 42 21 Saline34 (dosage N/A).

Example 35 Gapped Oligomeric Compounds Targeted to PTEN In Vivo Study

Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected once with the gapped oligomeric compounds targeted to PTEN at adose of 3.2, 10, 32 or 100 mg/kg. The mice were sacrificed 72 hoursfollowing administration. Liver tissues were homogenized and mRNA levelswere quantitated using real-time PCR as described herein for comparisonto untreated control levels (% UTC). Plasma chemistry analysis wascompleted. Tms were determined in 100 mM phosphate buffer, 0.1 mM EDTA,pH 7, at 260 nm using 4 μM of the modified oligomers listed below and 4μM of the complementary RNA TCAAGGCCAGTGCTAAGAGT (SEQ ID NO: 8) for2/14/2 motif oligomers and AGGCCAGTGCTAAG (SEQ ID NO: 7) for 2/10/2oligomers.

SEQ ID NO./ Composition Tm ISIS NO. (5′ to 3′) (° C.) Motif 03/411026C_(f)U_(f)GCTAGCCTCTGGATU_(f)U_(f) 57.1 2/14/2 04/418000^(Me)C_(f)T_(f)GCTAGCCTCTGGATT_(f)T_(f) 58.5 2/14/2 5-CH₃ wings01/410131 C_(f)U_(f)TAGCACTGGCC_(f)U_(f) 50.7 2/10/2 02/417999^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f) 52.6 2/10/2 5-CH₃ wings02/392063 ^(Me)C_(l)T_(l)TAGCACTGGC^(Me)C_(l)T_(l) 60.52/10/2 5-CH₃ wings

Each internucleoside linking group is a phosphorothioate and superscriptMe indicates that the following C is a 5-methyl C. Subscriptednucleosides are defined below wherein Bx is a heterocyclic base:

SEQ ID NO./ % UTC @ % UTC @ % UTC @ % UTC @ ISIS NO. 3.2 mg/kg 10 mg/kg32 mg/kg 100 mg/kg 02/392063 92 29 7 7 03/411026 92 52 12 7 04/418000100 38 12 5 01/410131 100 59 9 3 02/417999 94 31 10 5 Saline % UTC = 100

Liver transaminase levels, alanine aminotranferease (ALT) and aspartateaminotransferase (AST), in serum were also measured relative to salineinjected mice. The approximate liver transaminase levels are listed inthe table below.

SEQ ID NO./ AST @ AST @ AST @ AST @ ISIS NO. 3.2 mg/kg 10 mg/kg 32 mg/kg100 mg/kg 02/392063 57 86 81 27399 03/411026 166 78 69 130 04/418000 9094 80 345 01/410131 48 87 187 51 02/417999 72 126 99 55 SEQ ID NO./ ALT@ ALT @ ALT @ ALT @ ISIS NO. 3.2 mg/kg 10 mg/kg 32 mg/kg 100 mg/kg02/392063 9 13 10 18670 03/411026 25 20 26 115 04/418000 17 33 44 32101/410131 14 15 22 11 02/417999 13 22 15 11.

Example 36 Gapped Oligomeric Compounds Targeted to PTEN In Vivo Study

Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected once with the gapped oligomeric compounds targeted to PTEN at adose of 3.2, 10, 32 or 100 mg/kg. The mice were sacrificed 72 hoursfollowing last administration. Liver tissues were homogenized and mRNAlevels were quantitated using real-time PCR as described herein forcomparison to untreated control levels (% UTC). Estimated ED₅₀concentrations for each oligomeric compound were calculated usingGraphpad Prism as shown below.

SEQ ID NO./ Composition ED₅₀ ISIS NO. (5′ to 3′) (mg/kg) 02/417999^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f) 7.5 02/425857^(Me)C_(h)T_(h)TAGCACTGGC^(Me)C_(h)T_(h) 14.5

Each internucleoside linking group is a phosphorothioate and superscriptMe indicates that the following C is a 5-methyl C. Subscriptednucleosides are defined below wherein Bx is a heterocyclic base:

SEQ ID NO./ % UTC at dosage ISIS NO. 3.2 mg/kg 10 mg/kg 32 mg/kg 100mg/kg 02/417999 77 41 9 5 02/425857 76 72 20 6 Saline 100

Liver transaminase levels, alanine aminotranferease (ALT) and aspartateaminotransferase (AST), in serum were also measured relative to salineinjected mice. The approximate liver transaminase levels are listed inthe table below.

SEQ ID NO./ ISIS NO. 3.2 mg/kg 10 mg/kg 32 mg/kg 100 mg/kg AST (IU/L) atdosage 02/417999 72 126 99 55 02/425857 88 64 77 46 Saline 77 (dosage:n/a) ALT (IU/L) at dosage 02/417999 26 24 19 31 02/425857 28 26 29 51Saline 31 (dosage: n/a).

Example 37 Gapped Oligomeric Compounds

Oligomeric compounds were prepared having a gapped motif with variousgap and wing sizes. Tms were determined in 100 mM phosphate buffer, 0.1mM EDTA, pH 7, at 260 nm using 4 μM of the modified oligomers listedbelow and 4 μM of either the complementary RNA TCAAGGCCAGTGCTAAGAGT (SEQID NO: 8) for Tm¹ or AGGCCAGTGCTAAG (SEQ ID NO: 7) for Tm².

SEQ ID NO./ Composition Tm¹ Gapmer ISIS NO. (5′ to 3′) (° C.) Design02/417999 ^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f) 59.4 2-10-2 02/425858^(Me)C_(f)T_(f)T_(f)AGCACTGG^(Me)C_(f) ^(Me)C_(f)T_(f) 67.4 3-8-305/425859 T_(f) ^(Me)C_(f)T_(f)TAGCACTGGC^(Me)C_(f)T_(f)T_(f) 65.03-10-3 05/425860 T_(f) ^(Me)C_(f)T_(f)T_(f)AGCACTGG^(Me)C_(f)^(Me)C_(f)T_(f)T_(f) 70.4 4-8-4 06/425861 ^(Me)C_(f)T_(f)^(Me)C_(f)T_(f)T_(f)AGCACTGG^(Me)C_(f) ^(Me)C_(f)T_(f)T_(f) 74.3 5-8-4

Each internucleoside linking group is a phosphorothioate and superscriptMe indicates that the following C is a 5-methyl C. Subscriptednucleoside is defined below wherein Bx is a heterocyclic base:

Example 38 Hemimers Targeted to PTEN In Vivo Study

Six week old Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected once with the gapped oligomeric compounds targeted to PTEN at adose of 1.6, 5, 16 or 50 mg/kg. The mice were sacrificed 72 hoursfollowing last administration. Liver tissues were homogenized and mRNAlevels were quantitated using real-time PCR as described herein forcomparison to untreated control levels (% UTC). Estimated ED₅₀concentrations for each oligomeric compound were calculated usingGraphpad Prism as shown below. Tms were determined in 100 mM phosphatebuffer, 0.1 mM EDTA, pH 7, at 260 nm using 4 μM of the modifiedoligomers listed below and 4 μM of either the complementary RNATCAAGGCCAGTGCTAAGAGT (SEQ ID NO: 8) for Tm¹ or AGGCCAGTGCTAAG (SEQ IDNO: 7) for Tm².

SEQ ID NO./ Composition ISIS NO. (5′ to 3′) Tm¹ Tm² 02/412471^(Me)C_(l)T_(l)T_(l)AGCACTGGC^(Me)CT 65.5 62.5 02/429495^(Me)C_(f)T_(f)T_(f)AGCACTGGC^(Me)CT 63.8 59.6

Each internucleoside linking group is a phosphorothioate and superscriptMe indicates that the following C is a 5-methyl C. Subscriptednucleosides are defined below wherein Bx is a heterocyclic base:

SEQ ID NO./ % UTC at dosage ISIS NO. 1.6 mg/kg 5 mg/kg 16 mg/kg 50 mg/kg02/412471 85 51 20 23 02/429495 90 79 40 17 Saline % UTC = 100

Liver transaminase levels, alanine aminotranferease (ALT) and aspartateaminotransferase (AST), in serum were also measured relative to salineinjected mice. The approximate liver transaminase levels are listed inthe table below.

SEQ ID NO./ ISIS NO. 1.6 mg/kg 5 mg/kg 16 mg/kg 50 mg/kg AST (IU/L) atdosage 02/412471 67 67 69 4572 02/429495 95 54 77 58 Saline 68 (dosage:n/a) ALT (IU/L) at dosage 02/412471 29 31 33 3419 02/429495 33 31 38 23Saline 35 (dosage: n/a).

What is claimed is:
 1. A gapped oligomeric compound comprising at leastone tetrahydropyran nucleoside analog of the formula:

wherein independently for each tetrahydropyran nucleoside analog of saidformula: Bx is a heterocyclic base moiety; T₃ and T₄ are each,independently, an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound or one ofT₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the oligomeric compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup or a 5′ or 3′-terminal group; and wherein said gapped oligomericcompound is an antisense compound comprising a first region of from 1 toabout 5 contiguous monomer subunits a second region of from 1 to about 5contiguous monomer subunits and a third region located between the firstand second region comprising from 6 to about 14 monomer subunits whereineach monomer subunit in the first and second region is, independently, amodified nucleoside and each monomer subunit in the third region is,independently, a nucleoside or a modified nucleoside other than themodified nucleosides in the first and second region wherein at least onemodified nucleoside in the first and second region is a tetrahydropyrannucleoside analog of said formula.
 2. The gapped oligomeric compound ofclaim 1 wherein essentially each internucleoside linking group is aphosphorothioate internucleoside linking group.
 3. The gapped oligomericcompound of claim 1 wherein essentially each internucleoside linkinggroup is a phosphodiester internucleoside linking group.
 4. The gappedoligomeric compound of claim 1 wherein each internucleoside linkinggroup is independently a phosphodiester or phosphorothioateinternucleoside linking group.
 5. The gapped oligomeric compound ofclaim 1 wherein each monomer subunit in the third region is aβ-D-2′-deoxyribonucleoside.
 6. The gapped oligomeric compound of claim 5wherein at least one β-D-2′-deoxyribonucleoside is linked to atetrahydropyran nucleoside analog by a phosphorothioate internucleosidelinking group.
 7. The gapped oligomeric compound of claim 5 wherein thethird region comprises from 10 to 14 β-D-2′-deoxyribonucleosides.
 8. Thegapped oligomeric compound of claim 1 wherein the first and secondregion each have 2 contiguous tetrahydropyran nucleoside analogs of saidformula and the third region has 10 β-D-2′-deoxyribonucleosides.
 9. Thegapped oligomeric compound of claim 1 wherein the first and third regioneach have from 2 to 5 contiguous modified nucleosides and the thirdregion has 8 to 14 β-D-2′-deoxyribonucleosides.
 10. The gappedoligomeric compound of claim 1 wherein the first and second regioncomprise modified nucleosides independently selected from bicyclicnucleosides, 2′-modified nucleosides, 4′-thio modified nucleosides and4′-thio-2′-modified nucleosides.
 11. The gapped oligomeric compound ofclaim 10 wherein the first and second region comprise 2′-modifiednucleosides wherein each 2′-substituent is independently selected from2′-F, 2′-OCH₃ and 2′-O(CH₂)₂—OCH₃.
 12. The gapped oligomeric compound ofclaim 1 wherein each monomer subunit in the third region is aβ-D-2′-deoxyribonucleoside.
 13. The gapped oligomeric compound of claim1 wherein each pyrimidine heterocyclic base optionally includes a5-methyl group.
 14. The gapped oligomeric compound of claim 1 comprisingfrom about 10 to about 21 monomer subunits.
 15. The gapped oligomericcompound of claim 1 comprising from about 12 to about 17 monomersubunits.
 16. The gapped oligomeric compound of claim 1 comprising fromabout 13 to about 16 monomer subunits.